MP-96manual by girlbanks

VIEWS: 15 PAGES: 28

									MINI-PREP 96™
  New Model

Instruction Manual




  6195 Cornerstone Court East
     San Diego, CA 92121
        1-800-466-7949
         858-452-2603

 E-mail macres@macconnell.com
      www.macconnell.com

               
                                                 Table of Contents




A. Unpacking and Setting up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .          3-4

B. Guidelines for Plasmid Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .           5-7

C. Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .         8-
        Well Cassettes                                                                                           8-9
       4 Well Cassettes                                                                                           9-

D. Fluorescent Sequencing of Mini-Prep 96 Purified DNA . . . . . . . . . . . . . . . . . . . . . . . . . 13

E. Introduction to Method     . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .      4

F. Separation Process Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .          5

G. Cleaning, Over-Current, High Temperature Warning            . . .. . . . . . . . . . . . . . . . . . . . .      6

H. Troubleshooting      . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    7-8

I. Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    19

J. Appendix
        A. Screen Flow Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .            0 -
        B. User Programming Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .           -3
        C. Procedure for Lysis and Loading Samples with Lysis Buffers . . . . . . . . . . . . . . . .               4
        D. Midi-Preps (Mid-Scale Preps) on the Mini-Prep Instrument . . . . . . . . . . . . . . . .                 4
        E. PCR Band purification on the Mini-Prep 96 . . . . . . . . . . . . . . . . . . . . . . . . . .            24
        F. Genomic DNA Purification (Technical Note) . . . . . . . . . . . . . . . . . . . . . . . . . .          . 25


K. Price List for Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .          6

         Version 00705w




                                                           
A. Unpacking and Setting Up

The shipping box contains the following:
                          The Mini-Prep 96 instrument
                          Power cord
                          Instruction manual,warranty card, and instructional CD-ROM
                          Extra rig filters

         Carefully remove the Mini-Prep 96 instrument from its shipping foam and carton.

The Mini-Prep 96 should be installed on a level work surface (preferably near a sink) to accommodate the discharge
of electrophoresis buffer liquid. Alternatively, a discard vessel such as a 2 or 4 liter Erlenmeyer flask can be used if
a sink is not available.

Three lengths of tubing are located on the rear of the instrument:
                  a. The NEW BUFFER inlet tubing
                  b. The USED BUFFER outlet tubing
                  c. The OVERFLOW outlet tubing

Note: If the Automatic Bleach/Buffer Diluter (ABD-101) is to be installed, follow the steps in this box.
Set-up Connections:
a. Shorten the inlet tubing on the MP-96 to approximately  inches, then connect it to the hose barb on the ABD-
0 unit labeled “ to instrument.”
b. Connect the supplied /4” inside diameter tubing to the hose barb on the ABD-0 unit labeled “to reservoir,”
then place the other end of the tubing into the pure water reservoir.
c. Connect the six-prong electrical cable to the outlet on the rear panel of the MP-96 labeled
“ Automatic buffer diluter.”
d. Fill the bleach bottle with household liquid bleach, fill the buffer bottle with 100X buffer, place bottles in ABD-
0 unit and screw on lids with tubing inserted thru the lids and extended to the bottom of the bottles.
Prime tubes (only required when setting up instrument or when refilling bleach/buffer bottles)
e. Place ~ 2 liters of de-ionized water in the reservoir and turn on the instrument. Wait for the first menu screen,
press “Interrupt,” then press “B” to get to the second menu. Select “fill rig” and “bleach,” then press “Start” to al-
low bleach bottle tubing to fill (prime). Press “Interrupt” again, then “B” to get to the second menu. Select “fill rig”
and “buffer,” then press “Start” to allow buffer bottle tubing to fill (prime). Press “Interrupt,” followed by “Drain,”
to drain liquid from the rig before beginning a run.

If the instrument is to be used without the Automatic Bleach/Buffer Diluter unit, follow the steps below.

. The USED BUFFER OUTLET tubing should be placed where it can drain used running buffer into a sink. Ad-
ditional 1/4 inch (ID) tubing may be added to this line in order to reach a sink drain. Alternatively, a flask or other
container may be used to capture used buffer. Notice that the Used Buffer Outlet tubing has an anti-siphon check
valve in-line to prevent used buffer from flowing back into the instrument. The OVERFLOW OUTLET tubing is
not normally used in operation of the instrument.

2. Since the instrument is shipped without buffer in its inlet tubing, the pump may have difficulty priming for the
first run. On the first run, it may be necessary to briefly lower the inlet tubing closer to the work surface by tilting
the buffer reservoir to allow for pump priming.

3. Check the two electrophoresis Rig Filters to ensure that they are properly installed into the right corners of the
electrophoresis rig (see Figure B). A piece of white nylon sponge should be visible in the corner of the rig where
the drain basin meets the rig wall.




                                                             3
                                                             Figure 1A Front Instrument Components                                         Seet-
                                                                                                                                           ing Up




                                                                                                                                          Setting Up
                                                                   Fill/Drain               Float Switch

                                                                       Port                 temperature sensor


                                                                                   Cam shafts and cams
                                                                                                Electrode plugs
                                                                                                                       Fill/Drain Port

                                                                                                               Note: Two Rig filters
                                                                                                               should be lodged in the
                                                                                                               corner drain basin at all
                                                                                                               times during run.



                 Lid Switches
           Figure 1B
      Electrophoresis Rig
                                                                                                                 Cassette
                                                                                                                 Holders


          Figure 1C
    Automatic Buffer/Bleach
           Diluter
                                                                                          Figure 2: Rear Panel
                                            Automatic

                                                                                              Components               On/Off Switch
                                             buffer

                                             diluter

                          Bleach              outlet
 Vent Holes

                 Buffer                                Exhaust Fan
                                                                             Macconnell Research
                                                                                 Ser. #
                                                                                                                                   100-120 V 

                                                                                                                                   Power Inlet
                                                      New Buffer
                                                      Inlet Hose
                                   Connect

                                   to reservoir
                                                                                                                                   250 V, 3A fuse

                                                                     Used Buffer                                           250V, 5A fuse
                                                                     Outlet Hose
                                                                                                  Automatic
      Computer 

                               Bleach
                                                            Buffer

                                                                                     Overflow                     Interface Port
   Connect
     Buffer
                                            Check Valve
                                                                                    Outlet tubing Diluter Outlet

to instrument
                                                              4
B. Guidelines for Plasmid Growth                                                                                        Seet-
                                                                                                                        ing Up
                 ESSENTIAL FOR GETTING GOOD YIELDS




                                                                                                                       Conditions
The Mini-Prep 96 is designed to purify plasmid DNA directly from bacterial cultures of E. coli. Its yield is greatly




                                                                                                                       Growth
affected by the amount of plasmid actually present in the E. coli culture. We have found that growth conditions of
E. coli vary greatly depending on:

                  ) Proper aeration of the culture
                  ) Shake rate
                  3) Temperature of growth
                  4) Length of time of growth
                  5) The growth medium
                  6) Age of the streak/colony used to initiate a culture
                  7) Sterility of the culture
                  8) Copy number of the plasmid

The Mini-Prep 96™ separation process is sensitive to changes in growth conditions. Growth conditions that yield
DNA with other purification kits and protocols may not give optimal results with this instrument.

1. Recommended Growth Conditions:
       a. Temperature/Time: 37˚ ± 1˚ C for 20 hours. Shorter growth time with MR-2001 will
               give lower yields. Note: MR-2001 does not contain anti-biotic when shipped.
       b. Shake Rate: 00-30 RPM in a gyratory environmental shaker
       c. Type of container and volume per flask:
               Always use one of the types of growth vessels with volumes as follows:
               . Round bottom disposable glass culture tubes (16 X 150 mm) with 2 ml or less of culture
               2. Falcon 2059 or 2057 snap cap tubes (17 X 100 mm) with 2 ml or less of culture
               3. Erlenmeyer flask with 20 - 50 ml per 250 ml
               4. Deep well microtiter plate growth blocks (with 2ml square wells) containing only 1 ml
               of culture. Use the Mini-Grow 384 microtiter block shaker at a vigorous RPM to shake these
               deep well blocks. We recommend sealing the top of the blocks with lab tape, then pricking
               holes in the tape to allow aeration during growth. Alternatively, microporous tape is availabe
               from MacConnell Research (Cat# MPT-00).

      THE VOLUME OF CULTURE PER GROWTH TUBE OR FLASK IS CRITICAL TO GETTING
GOOD YIELDS WITH THE MINI-PREP 96.

In the data shown below, volumes ranging from  to 0 ml of culture were inoculated and grown identically in 5
ml (16 X 150 mm) glass disposable culture tubes for 20 hours at 37ºC. One ml of each culture was processed in a
2.0 ml cassette with the Mini-Prep 96 instrument. The resulting purified plasmid DNA was run on a 1% agarose gel.
The yield rapidly declines with an increase of culture volume per tube.  mls per tube gave optimal yields.

                                                                  tube       3   4   5   7 0




                                                           5
Figure 3. Recommended Culture Growth Tubes:                                                                               Seet-
                                                                                                                          ing Up
         Best              Good                                Good                                   Bad




                                                                                                                     Conditions
Glass 16 X 150 mm
                               96 Deep well plate with 1 ml 
                      Screw Cap





                                                                                                                     Growth
   growth tube            17 X 100 mm                                                                15 ml vial
                                                 of culture in 2 ml square wells.

                        Falcon 2059/2057
                           
                     Grow plate in Mini-Grow 384
                           Snap-Cap              shaker




Other factors:
         The following strains of E. coli are recommended:
                                                      Yield with .0 ml of Bluescript/DH5a
                  E. coli cell line                       with short body cassettes
                  . DH0B                                    3-6 mg
                  . DH5aF’                                   3-6 mg
                  3. DH5a                                     3-6 mg
                  4. DH5                                      3-6 mg
                  5. JM09                                    -6 mg
                  6. HB0                                    3-5 mg
                  7. LE39                                    3-6 mg
                  8. MC06                                   3-6 mg
                  9. INV1aF’                                  3-6 mg
                  10. INV1a                                   3-6 mg
                  . SURE                                    -3 mg
                  . DHS                                   3-6 mg
                  3. TOP 0F’                                3-6 mg
                  4. TOP 0                                  3-6 mg
                  15. XL-1 Blue (1994)                        3-6 mg
                  6. SOLR                                    0. mg
                  7. RR                                      mg
                  8. BB4                                     -6 mg
                  * All DNA > 95% supercoiled


Some bacterial strains contain nucleases that degrade plasmid DNA during the Mini-Prep 96™ electrophoretic
process. In general, these nucleases are excreted into the growth media and can be removed from the culture by
concentrating the bacterial cells using centrifugation. Since the Mini-Prep 96™ allows you to load bacterial culture
directly, we recommend the use of the high yielding strains shown in the previous table.
2. Growth Medium

We strongly recommend the use of Magnificent Broth (MR2001) with the Mini-Prep 96 process. MR2001 has
several features that result in maximal yields with the instrument process: () MR00 is buffered so that even after
growth of up to 4 hours, the culture is still at pH 7.8 to 8.0, which is compatible with the pH of the running buffer,


                                                         6
(2) MR2001 has low salt content (i.e. less than 0.5 gm/liter of NaCl), which does not interfere with the separation        Seet-
process, and (3) MR00 will allow the plasmid copy number to increase after the bacterial cells have reached a
                                                                                                                           ing Up
steady state in their growth cycle. This media produces higher DNA yields per volume than other media for this
instrument. It will also produce excellent yields with other mini-prep protocols.




                                                                                                                       Conditions
Up to 5 ml of bacteria can be concentrated for loading into one cassette lane. Increasing the amount of bacterial




                                                                                                                       Growth
cells will raise the yield up to three times that obtained from  ml.

Growth media that give reasonable yields when cells are pelleted prior to addition of lysis buffers include: L-Broth,
YT Broth, 2X YT Broth, and Super Broth. Terrific Broth does not provide good yields and will cause an overcur-
rent condition to occur with the instrument.

3. Age of the Bacterial Colony or Streak

Begin culture inoculation with bacterial colonies or streaks that are less than two weeks old. The age of a bacterial
colony or streak used to inoculate a liquid culture will affect the plasmid yield. Inoculations of a culture from a fro-
zen glycerol stock will generally result in lower yields when compared to inoculation from fresh bacterial colonies.
Never innoculate a liquid culture from another liquid culture.

4. Sterility of the Culture

Inoculate cultures using sterile techniques. Antibiotics should be used at all stages of E.coli growth. The Mini-Prep
96™ purification procedure can be adversely affected by the sterility of the culture. A culture infected with foreign
organisms will typically result in low yield and/or degraded plasmid DNA due to the presence of bacterial nucleases
that arise from these foreign organisms.

5. Plasmid copy number

We recommend the use of high copy number vectors as results obtained with low and medium copy number vectors
can be low. The copy number of a plasmid in E. coli depends on: replication origin (ColE, pMB or pSC0),
insert size, and type of insert, among other factors. In general, plasmids can be grouped into three copy number cat-
egories: i) high copy number, such as pUC, pBluescript and their derivatives (300-650 copies per cell), ii) medium
copy number, such as pBR3, pACYC, cosmids, etc. (0-0 copies per cell), and iii) low copy number, such as
pSC0 and other single copy number vectors. In addition to the above, there are single copy large plasmids, such
as PACs, YACs, and BACs that are used in molecular biology research. Refer to the subsequent procedures for a
method to spin down and load 5-0 ml of cells for medium copy number vectors.




                                                            7
C. Operation
                                                                                                                     Seet-
The following items are required for purifying DNA from the Mini-Prep 96 instrument:                                 ing Up

                   Cassettes (provided with Starter Kit)




                                                                                                                     Operation
                  Applicator Comb and Lysis Tablets (provided with Starter Kit or purchased cassettes)
                           (or Lysis Buffers, if ordered)
                  100X Running Buffer (provided with Starter Kit)
                  Running Buffer reservoir (2 liter Erlenmeyer flask)
                  Used Buffer reservoir or sink/drain

         Additional supplies recommended for the procedure:

                  0-00 ul and 0-000 ul pipettors (-channel pipettor recommended) and tips
                  5 or 0 ml serological pipette
                  Sharp scissors
                  Household liquid bleach

 It is most efficient to load the sample cassettes with culture outside the instrument prior to performing the bleach,
rinse, and running buffer preparation steps (See steps 4 & 5 on next page). This sequence of steps allows the culture
to incubate with the reagents contained in the Applicator Comb/Lysis Tablets for at least the time needed to bleach
and rinse the instrument, thus enhancing the lysis of bacteria and increasing the yield.

Using 12-Well Cassettes
Step 1. Turn the instrument on. Remove a disposable cassette from its storage pouch and slide the microchamber
block upward until its lower surface is just above the level of the gel medium in the cassette. Make sure that the
raised microchamber block is parallel to the cassette floor (Figure 4). This will prevent the block from jamming
when it is activated by the rotating cams.




     Figure 4 Microchamber Block Position

                                               Sample Wells

                                                            Finished 

                                                            Sample Wells




                                       Slide Microchamber

                                       Block upward above 

                                       gel level of the cassette
                                       before starting.



                                                        8
   Figure 5A. Procedure for Using the                                                                                        Seet-
   Applicator Comb/Lysis Tablets                             Step 2. Remove the Applicator Comb from its bag and
                                                                                                                             ing Up
                                                             place in sample wells. Pipet bacterial culture into the
                      Micro-                                 sample wells (we recommend .5 -  mls of culture).
                     chamber
           Insert
              Swish Applicator Comb back and forth a few times.




                                                                                                                             Operation
                   Block in the 
       Applicator
                    After approximately one minute, invert Lysis Tab-
                  "up" position         Comb
                                                             let Dispensor over sample wells of cassette. Remove slider
                                                             to one side allowing the tablets to fall into the individual
                                                             wells. Swish the Applicator Comb back and forth a few
                                                             times to stir the wells.
                                                                       Allow the culture to incubate with the Applicator
                                                             Combs and dissolved Lysis Tablets while performing the
                                                             steps below. White powder binder from the lysis tablets
                                                             will remain undissolved in the well bottoms. See Fig. 5A.
                                                                       The display will prompt you to set the lysis timer
                                                             at this point (5 minutes is the default and a typical lysis
                                               Applicator

                                                 Comb        time.). This will be the amount of time that the instrument
Pipet bacterial culture 
                                    waits before it begins the automatic run once the program
  into sample wells.
                                        is selected in Step 8 below. The lysis timer can be adjusted
                                                             using the arrow keys to lengthen or shorten the lysis time.
                                                             Do not place cassette(s) in rig until step three has been
                                                             completed.

                                                             Using the 24-Well Cassettes
                                                                       24 well cassettes fit into the instrument and run
                                                             on program 4. The microchamber block of the 4-well
                                                             cassette is permanently fixed in the down position and,
                                                             therefore does not move during the run. 4 –well cassettes
                                                             hold only  ml of culture per lane and are supplied with
                                                             liquid lysis buffers in place of applicator combs and Lysis
                                                             Tablets. The liquid lysis buffers are added to the cassette
                                                             wells using a -channel pipettor in every other lane per
       Invert

    Lysis Tablet 
                                           load. Liquid lysis buffers should be thawed and poured
   dispensor over                                            into a clean reagent reservoir that is provided with 4-well
    sample wells
            Tablet                          cassettes. The yield of the 4 well cassette is typically
                            Dispensor
                                                             /3 that of  well version. Load each lane of the 4-well
                                                             cassette with culture using a single-channel or -channel
                                                             pipettor. In order add 00 ul of Lysis Buffer A (stir with
                                                              channel pipettor tips) then add 0 ul of Lysis Buffer B to
                                                             each lane from separate reagent reservoirs. Stir the Buffer
                                                             B into the culture using the multi-channel pipettor. A num-
                                                             bered loading template is provided in each pack of cassettes
                                                             that helps identify lanes during loading and un-loading.
                                                             Note: Program 4 is the optimal program for running the
                                                             4-well cassettes and will also work with -well cassettes.

                                                             Step 3. While the cultures are lysing in the cassette wells,
                                                             perform the recommended bleach cycle by carrying out the
                                                             following steps as prompted by the instrument’s display.
                                                             Cassettes should not be in rig during the bleach cycle.
                                                             This step should be carried out prior to the first run of
                                                             each day.



                                                             9
                                                                                                    Reagent                  Seet-
                                                                                                    reservoir                ing Up




                                                                                                                             Operation
                                   24-well cassette
Note: If the Automatic Bleach/Buffer Diluter (ABD-101) is attached to your instrument, proceed to the the instruc-
tion in the box below. Otherwise follow steps a and b.
          (a) Place 1 liter of a ~2% bleach solution made with tap water in the new buffer reservoir or similar vessel
(0 mls of liquid household bleach and 980 ml of water). Without cassettes in the rig, press “start/enter” to draw the
bleach solution into the rig. The instrument will automatically fill, incubate for 20 seconds, then drain.
          (b) When prompted by the display, discard the remaining bleach solution in the New Buffer Reservoir and
replace it with 1 liter of de-ionized water. Press “Start/Enter” and the instrument will automatically fill itself with
this water and drain as before, effecting a rinsing step. The instrument is now ready for DNA purification.
The above bleaching procedure is not necessary between runs made in the same day; however, it is necessary to per-
form a water rinse step between runs. The instrument will prompt you to put pure water in the new buffer reservoir
after the run so that it can automatically rinse the rig.

If the Automatic Bleach/Buffer Diluter (ABD-0) is attached to your instrument and its tubings have been primed
(See page 3), simply place 2 liters of de-ionized water into the New Buffer Reservoir and proceed with the steps
indicated on the instrument’s display. Carry out all of the automatic bleach and rinse steps prompted by the display
until you come to the cassette insertion step.

Step 4. Lift the front end of the camshafts that sit above the instrument’s rig to move these shafts out of the way.
The front of these cams will snap loose from their fittings located on the inside front wall of the instrument. Insert
the loaded cassette(s) into the cassette holder slots in the empty electrophoresis rig by lining up the “feet” of the
cassettes in the cutout slots. Use the slots in the rig closest to the instrument’s metal wall if less than four cassettes
are processed. Be certain that the cassette(s) are positioned (as shown in figure 5B) between the white teflon hold-
ers and fully seated on the small platforms on the rig floor with the raised microchamber block(s) facing the rear
wall of the instrument. Snap the cam shafts back into place. The microchamber block(s) should then be directly
under the cam(s). (Figure 5B). The sample wells for either the  well or 4-well cassette should be nearest to you.

Figure 5B. Inserting Cassette(s) into Rig (Sample Wells Must Be Nearest to You)


                                                        Cam shaft

                                                                        Cassette
            Float switch




                                                                              Rig front

                                                               Cassette holder slot

                                                         Cam shaft snap-in fitting

                                                          0
If the Automatic Bleach/Buffer Diluter (ABD-0) is attached to your instrument, place  liters of high purity water
                                                                                                                          Seet-
into the New Buffer Reservoir, then proceed with the steps prompted by the instrument’s display to fill the rig (cas-     ing Up
settes in place) with 1X Running Buffer. Otherwise follow the steps below.




                                                                                                                          Operation
Step 5. Prepare the working concentration of electrophoresis Running Buffer (RB). We recommend 000 mls per
run. Dilute the stock container of 100X RB as follows:
Put 20 mls 100X RB into ~2000 mls of NANOpure, Milli-Q, or equivalent water, then mix. The working con-
centration of running buffer must be made fresh for each run. DO NOT STORE DILUTED RUNNING BUFFER
OVERNIGHT; IT WILL BREAK DOWN. (100X Running Buffer is stable indefinitely).

Step 6. Place the end of the NEW BUFFER INLET tubing into the running buffer. Be sure that the USED BUF-
FER OUTLET tubing goes to the empty discard reservoir or a sink.

Step 7. Make sure that lysis has occurred, as evidenced by an increase in viscosity of the culture when pipet-
ted up and down. If problems with lysis occur, incubation at 37ºC will speed up the lysis process. Leave the
Applicator Comb in the cassette throughout the run.

Step 8. SELECT PROGRAM
          P1       Standard plasmid purification program for C1202-SB (or C2402-SB) cassettes (70 minutes)
          P2       Short program for plasmid purification with C1202-SB cassettes (60 minutes)
          P3       Program for large plasmids (> 5kb) with C1202-SB cassettes (70 minutes)
          P4       Program for the 4-well cassettes
          G1       Program for genomic DNA purification (90 minutes)
          U       User programming feature for custom run programs (see Appendix B)
After selecting the appropriate program, the instrument will wait for the remaining time on the Lysis Timer, then
automatically fill with 1X Running Buffer and begin the DNA separation process. No further user attention will
be required until the end of the run. The total time remaining in the run is continually displayed on the instrument
readout panel, along with the rig buffer temperature and electrophoretic current. At the completion of the program,
the instrument will drain the running buffer and alert the user to remove the cassette(s) by emitting a beeping sound .
Step 9. At the end of the run, remove cassette(s) from the instrument and place on a paper towel with the micro-
chamber block facing toward you. Remove the 0-5 ul of sample from each microchamber block well with a 00
ul disposable-tip pipettor, or use an 8- channel multi-tip pipettor to remove samples simultaneously (Figure 6).
To get optimal yields from the instrument, you must remove the samples from the microchamber block within fifteen
minutes of the end of the run.

Figure 6. Removing Purified DNA Sample from Cassette Microchamber Block


                                                            Pipet tip

                                                            in well


                                                                                                      Pipet tip
                                                                  Microchamber

                                                                          Block




                                         Purified DNA

                                   in ~25 ul in microchamber
                                         Membrane
                                 block wells next to membrane


                                                          
Figure 7. Removing Purified DNA Sample from a 24 Well Cassette
                                                                                                                 Seet-
                                                                                                                 ing Up




                                                                                                                 Operation
Samples can be removed from the 4 -well cassette using either a single-channel pipettor or -channel pipettor.
Since the -channel pipettor will remove every other lane, use the numbered loading template provided with your
cassette pack to keep track of sample numbers and lanes.
Prior to the second and any subsequent runs of the day, the instrument should be rinsed with de-ionized water. This
step will also be prompted by the display at the end of each run.

NOTES:
1. You may find that plasmid purified on the Mini-Prep 96 will have a slight yellow color. This yellow com-
pound absorbs UV at 260 nm, and therefore prevents the correct reading of DNA concentration by OD 260
readings. This compound is a vitamin that co-purifies with the DNA and is not inhibitory to any enzyme that
we know of, nor is it toxic to mammalian cells. It does not interfere with automated fluorescent DNA sequenc-
ing. To determine the concentraton of MP-96 or MP-24 purified DNA, we recommend the use fluorometry or
by analyzing the density of the ethidium bands on an agarose gel. Either of these methods can be perform on
as little as 2 ul of the purified DNA.

2. It is best to analyze purified DNA on agarose gels run in Tris-acetate-EDTA (TAE) buffer. Gels runs in TBE may
show the incorrect size of bands due to TAE-like buffer used in the Mini-Prep electrophoretic purification.
3. Any running buffer that remains in the electrophoresis rig after the run will become infected with bacteria if left
standing overnight. Bacterial infection will cause nuclease contamination in the next run, which can degrade the
DNA once it is in the presence of the enzyme buffers. Therefore, the instrument rig and tubing should be sterilized
prior to the first run of a day with a 2% bleach solution in warm water. The 2% bleach solution will rid the tub-
ing, rig, internal pump and valves of algae and bacteria, along with their associated nucleases and proteases. The
instrument’s display will prompt you to perform the bleach treatment when it is first turned on. It is important that
substantially all of the bleach and rinse water be drained from the instrument prior to use, as the residual bleach will
interfere with the separation of DNA.
4. You can stop the run at any time during the run process without the Mini-Prep 96 losing track of where its separa-
tion process left off. Simply press “Interrupt” to suspend the run. You can then press (a) “Drain” to terminate the
run and drain the buffer, (You can terminate the action of the pump at any time by pressing “Interrupt,” followed by
“Start/Enter,” to return to the beginning of the program.) (b) “Continue” to resume the run, (c) “Start/Enter” to start
the run over, or (d) the “A” or “B” key go to additional menus in the Interrupt mode.
5. The length of the draining pump time can be adjusted by pressing “B” in the interrupt screen.
Never Use Alcohols or Organic Solvents to Clean or Rinse the Electrophoresis Rig

                                                        
D. DNA Sequencing with Purified DNA
a. Fluorescent Automated Sequencing
          DNA purified from the Mini-Prep 96 is a good template for automated fluorescent sequencing using cycle
sequencing protocols. We have found that plasmid DNA taken directly from the instrument will give 800+ (base)
sequencing reads on ABI 377, 3100, 3700, and 3730 instruments with Big-Dye chemistries. The DNA taken di-
rectly from the MP96 will also sequence with Licor instruments.
          The following is a guide for the amount of Mini-Prep 24 or 96 purified DNA to use in the automated fluo-
rescent sequencing protocol.
             Automated Fluorescent Sequencing Signal Strength and Actual Read Length Examples
Nanograms of MP-96/24 purified (4 kb) plasmid DNA              Average Signal Strength             Read Length*
          00 ng                                                       400                       745
          900 ng                                                        340                       775
          600 ng                                                        30                        88
          300 ng                                                        890                       88
          40 ng                                                        40                       87
          60 ng                                                        300                       8
          80 ng                                                         57                        874
Nanograms of MP-24/96 purified (18kb) plasmid DNA              Average Signal Strength             Read Length
          50 ng                                                         300-450                    875
          50 ng                                                        350-500                    855
* Read length is the base number of the sequence read before the first “N” appears.
          Note: We have concluded from the above data that template quantities from 50-240 ng of purified plasmid
gave the best read lengths. Other data of this sort shows that 1-2 microliters of the purified DNA generally gives the
best quality sequence data, provided that the concentration of the purified DNA is at least greater than 0.04 ug/ul for
2 ul submitted DNA or 0.08 ug/ul for 1 ul of submitted DNA. We do not recommend using more than 3 ul of the
purified DNA in a sequencing reaction and, since the results show that less template gives better sequence reads, use
of less than 2 ul of the purified DNA, (or ideally 1 ul) gives best results. This is due to the fact that less electropho-
resis buffer salt is carried into the sequencing reactions with less volume of the purified DNA. DNA concentrations
should be determined by fluorometry or by running a 2 ul aliquot of the purified DNA on a 1% TAE-agarose gel.
CONCLUSION: Use 1-2 ul of the instrument purified DNA for automated fluorescent sequencing.

If automated fluorescent DNA sequencing does not work with DNA taken directly form the instrument, we recom-
mend the following protocol. Precipitate 20-25 ul of the DNA with 2 volumes of ethanol and 0.1 volume of 2 M
sodium acetate, then rinse this pellet with 70% ethanol prior to use in fluorescent sequencing protocols.

b. Manual Sequencing
          Plasmid DNA prepared on the Mini-Prep 96™ is highly active in manual DNA sequencing protocols and
gives good quality sequence data. The amount of DNA that is required for good manual sequencing results ranges
from 0.0 - 0.5 micrograms.
          When using alkaline denaturation prior to primer annealing, we suggest that you use one half of the amount
of salt in the ethanol precipitation step immediately following the alkaline denaturation step.
          A typical alkaline denaturation protocol is as follows:
                    0.25 - 0.7 ug of DNA in 18 ul. Water may be added to bring the DNA up to this volume.
                    add 2.0 ul of 2.0 M NaOH and incubate at 37ºC for 5 minutes
                    add 0. volumes of 7.5 M ammonium acetate solution (pH 7.5)
                    add  volumes of ethanol, place on ice for 5 minutes, spin for 0 minutes, wash
          with 70% ethanol/water, spin a second time, remove the traces of ethanol
Note: You may find that plasmid DNA purified from bacteria grown in MR-2001 and processed on the Mini-Prep
96 will have a slight yellow color. This yellow compound absorbs UV at 60 nm, and is visible as a light blue color
on agarose or acrylamide gels stained with ethidium bromide and viewed on a UV transilluminator. This compound
is a vitamin that co-purifies with the DNA and is not inhibitory to any enzyme that we know of, nor is it toxic to
mammalian cells. Yield measurements by OD260 will not be accurate.
c. Re-Purification of Plasmid DNA             DNA prepared by other protocols can be re-purified on the Mini-Prep
96. If DNA is re-purified with the Mini-Prep 96, follow the exact procedure, as is used with bacterial culture, except
omit the addition of the Applicator Comb, and add only the Lysis Tablets and DNA solution to the cassette.
                                                                       3
E. Introduction to Method


          The Mini-Prep 96™ is a fully automated, bench-top instrument designed for the purification of plasmid
DNA directly from bacterial culture. The instrument uses a revolutionary new method of nucleic acid purification
based on agarose gel electrophoresis and subsequent nucleic acid recovery by electroelution. The resulting DNA is
highly pure and can be used for DNA sequencing, restriction digests, transformations, transfections or as a substrate
for DNA modification enzymes.
          The separation process begins with freshly grown bacterial culture to which a lysis solution is added. The
lysed bacterial samples are loaded directly into a sample cassette, which is placed into the instrument for processing.
Alternatively, the lysis buffers can be mixed simultaneously during loading through the use of the Applicator Combs
and Lysis Tablets , which affects the addition of lysis reagents to the sample wells prior to the run.
          Microprocessor-controlled electrophoretic separation is performed automatically according to one of
several user-selected programs. The instrument automatically recovers the individual samples of purified DNA by
electroelution and concentrates them into microchambers, where they can be removed using a disposable tip micro-
pipettor.
          The instrument can process up to four cassettes in a run, with each cassette capable of holding up to 
(or 4) separate bacterial samples. Individual sample chambers within the C0-SB cassette hold up to .0 ml of
starting culture while wells of the C40-SB cassette hold up to .0 ml of starting culture, and typically yield -8
micrograms of purified plasmid DNA in ~ 25 ul volume. The entire purification process takes 60-75 minutes,
excluding time for loading and unloading the cassettes.

         Features:

         Automatic operation
         Begins with bacterial culture (no centrifugation steps)
         Yields highly pure DNA
         No phenol or chloroform
         Bench top size (4”w x 0 “ l x 9 “ h), < 5 lb.
         Economical
         Pure enough for manual and fluorescent automated sequencing straight from the instrument.
         Purification of various sizes of plasmid DNA is accomplished with the same program.
         DNA cuts to completion with common restriction enzymes, and can be used for trasnfection,
                  trasncription, ligation and transformation.
         Midi-Preps can be performed by loading multiple lanes or one or more cassette with the same culture.




                                                        4
F. Separation Process Description
      The instrument’s purification process involves four main steps as described below and diagrammed in below.
           () The bacteria are lysed directly in the culture media using a set of reagents that break open the bacteria,
degrade RNA, digest proteins and dissolve bacterial membranes.
            The lysis components referred to as Lysis Buffer A and B are packaged in two separate devices to prevent
degradation of their enzymes during storage. These are the Applicator Combs and Lysis Tablets. The Applicator
Comb has the Lysis Buffer A reagents absorbed into foam pads that are mounted on a disposable plastic comb. The
Lysis Tablets contain the enzyme components of Lysis Buffer B along with the pill binder. The Applicator Comb is
inserted by the operator into the wells of the cassette prior to sample loading. Use of the Applicator Comb and Lysis
Tablets, therefore, allows the culture medium to be loaded directly into the cassette wells prior to starting the run,
eliminating the mixing steps. This method requires that the bacterial cells be grown in MR00 medium. Alterna-
tively, Lysis Buffers A and B can be purchased in liquid form.
           (2) DNA and other solubilized molecules are resolved on an agarose gel that has been pre-cast by the
manufacturer into a disposable plastic cassette. The resolution process is accomplished through a microproces-
sor-controlled power supply and electrophoresis rig.
Plasmid DNA in the crude lysate is separated from the
degraded RNA, proteins, and bacterial chromatin DNA
by its unique mobility in agarose during programmed
electrophoresis. During the separation process, plasmid
DNA, ranging in size from 2-15 kb, is electrophoresed
until ~ kb super-coiled plasmids run to the end, but not
off, of the separation gel. At this point in the run the
electrophoresis running buffer is automatically changed
and the electroelution stage is commenced.
           The running buffer for the electrophoresis is
a derivative of Tris-acetate EDTA (TAE), where the
Tris-acetate concentration has been lowered to 0 mM
and the EDTA is present in trace amounts. The working
pH of this running buffer is 8.0, which matches the pH
of both the agarose separation medium and the bacterial
growth medium. All three of these components must
be at the same pH and relative ionic strength in order
for the separation process and voltage timing to work
properly.
           (3) Electroelution of the resolved DNA from
the gel is accomplished by a device containing a high
cut-off ultrafiltration membrane. This elution device, or
Microchamber Block, slides over the end of the separa-
tion gel and in register with each cassette lane. This
microchamber block is pushed into place by a motor-
driven camshaft and rotating cams that are activated by
the instrument’s computer at the end of the separation
stage of the run. During the elution stage, the DNA
collects as a film on the inner vertical surface of the
ultrafiltration membrane. The DNA remains in place
until the draining step occurs. In the 4 -well cassette
the microchamber block with ultra-filtration membrane
is actually permanetly fixed to the end of the purufcation cassette.
           (4) At the end of the electroelution process, the instrument automatically drains the running buffer from
its rig, which also causes the buffer to drain from the internal portion of the microchamber block. At this point, the
purified DNA loses its attraction to the membrane, due to the loss of electrophoretic conductivity in the rig, and falls
downward into the trapped droplet of buffer at the bottom corner of the microchamber block window. This droplet
with purified DNA ranges in volume from 20 -25 ml and is easily removed from the microchamber block using
a disposable tip pipettor. Since the finished sample wells of the microchamber block are spaced like a microtiter
plate, samples can be removed simultaneously using a multi-channel pipettor.
                                                           5
G. Cleaning
           A. Although it is rarely necessary, the electrophoresis rig can be removed from the instrument for cleaning
by detaching its (1) float switch plug-in jack , (2) electrode connectors, and (3) tubing fitting. It is also necessary to
lift the cam shafts out of the way to remove the rig. Clean the rig with warm water containing ordinary detergent
and rinse thoroughly with de-ionized water before reinstallation. Over time, the float valve can become coated with
a film which can result in the instrument overfilling its electrophoresis rig. It is advisable to rinse and wipe the float
valve when the rig is removed for cleaning. Never clean the rig with ethanol or any other organic solvent.

          B. The electrophoresis rig has two small plugs of nylon mesh in its upper right rig corners that act as filters
for fragments of agarose and other particulate materials that might become drawn into the pump and valves during
the drain and rinse cycles. This nylon mesh filter needs replacement or cleaning when the pumping rate during the
draining cycle appears slower than usual or when the rig will not fully drain. This filter can be removed by using a
plastic pipet tip or a hemostat. We advise cleaning the filters after every fourth run to avoid problems.

         C. We recommend that the instrument be treated with a 2% bleach solution before the first run of each
day using the procedure described on page 8. This treatment will rid the tubing, rig, internal pump and valves of
algae and bacteria that secrete nucleases that degrade the purified DNA. This step can be accomplished by pumping
warm water containing 2% bleach into the instrument’s rig (without cassettes) when prompted at the beginning of
the run program. Simply fill the new buffer reservoir with ~ 1 liter of 2% bleach and press “Start/Enter”. Once the
instrument has filled with the bleach solution, wait ~10 seconds, then drain this solution out by pressing “Interrupt,”
followed by “Drain”. The bleach-treated tubing and valves should then be rinsed in the same way with one or two
passes of de-ionized water prior to subsequent use.

        D. When transporting or shipping the instrument, drain all of the fluid out of the inlet and outlet tubing by
executing the filling and draining steps with an empty reservoir.

Over-Current Condition
          The instrument will automatically lower its electrophoresis voltage if the current is too high for the power
supply’s specification. The instrument will indicate this condition along with the time remaining on its display.
Over-current is almost always caused by the use of growth media that contains more than 0.5 gm/liter of salt. This
salt electrophoreses into the Running Buffer during the run and causes an increase in current, which can overload
the power supply. Over-current can also be caused by running buffer that is made more concentrated than 1X. If
you get an over-current diagnostic on the display, you must terminate the run and change the running buffer prior to
continuing or restarting the program. After this buffer change, the remainder of the run can be programmed by the
operator after noting the time remaining displayed on the over-current screen. Changing the buffer, however, will
cause some yield loss due to the draining event.
           Call MacConnell Research to get advice on finishing the run by programming the remaining step(s).

High Temperature Warning
          The instrument has a temperature sensor probe adjacent to its float switch. This device communicates to
the instrument’s computer, which displays the temperature of the running buffer on the LCD during the run. If the
running buffer temperature exceeds 37º C, the instrument will display a warning message and automatically lower
it electrophoresis voltage. Temperatures above 37º C will melt the agarose in the cassette(s) and result in impure
DNA. High temperature is almost always caused by salt ions in the running buffer that come from improper culture
media or incorrectly made running buffer. Room temperatures above 75 º F can also caused the high temperature
condition. The instrument will continue its automatic run even with a High Temperature Warning.




                                                         6
H. Trouble Shooting Guide                      SUGGESTED SOLUTION
        PROBLEM
                                               A. Have the growth conditions described in the manual been
1. DNA yield is too low
                                               followed, including: growth time of 8-0 hours, limited culture
                                               volume per flask/tube, sterile growth conditions, innoculation
                                               from a single colony off a plate? The MP-96/4 procdure is more
                                               sensitive to growth conditions than prep kits.
                                               B. Is there lysis of bacterial culture ~ 0-5 min. after loading
                                               over Applicator comb and Tablets ( i.e. does the culture become
                                               viscous)?
                                               NO: i. Freeze the lysis mixture in dry ice or a freezer, thaw and
                                               reload. Lysis reagent should be stored at -20ºC prior to use.
                                               ii. If culture will not lyse, start with new culture, spin down
                                               culture and resuspend in 1.0 ml of 1X Running Buffer or water
                                               prior to loading.
                                               YES: culture becomes viscous ~10 min. after loading. (Note:
                                               always wait 30 or more seconds after loading the culture over the
                                               Applicator combs before adding the Lysis Tablets.)
                                               i. Strain may be contaminated with foreign bacteria. Streak the
                                               bacterial strain out on an antibiotic plate and grow from a single
                                               colony.
                                               ii. Strain is old. Streak the strain out on an antibiotic plate and
                                               grow from single colony.
                                               iii. Strain may have a nuclease that is secreted into the growth
                                               media. Spin down -5 mls and resuspend pellet in 0.5 mls prior
                                               to start. For very low yielding cultures, concentrate up to 5 ml of
                                               culture by centrifugation and resuspend in 0.5 ml of 1X Run-
                                               ning Buffer prior to loading with combs and Lysis tablets.
                                               C. Do fine bubbles appear at the front platinum electrode in the
                                               rig after starting program run?
                                               YES: Try one of the above solutions.
                                               NO: Running Buffer is made incorrectly and power supply has
                                               shut off automatically. Press “Interrupt”, followed by “Drain”,
                                               and then wait for buffer to drain. Turn off instrument, wait ten
                                               seconds, remake running buffer and start the program over.

                                               Have you grown your culture in L-broth or other broth containing
                                               more than 0.5 gm/liter of salt?
                                               Yes: Spin down culture and resuspend in 0.5 ml 1X Running buf-
                                               fer prior to loading, or re-grow culture in MR00 broth.
                                               No: Try one of the above suggestions or call the company.




                                               A. This streak of material is degraded genomic DNA resulting
2. Purified DNA has a streak of degraded DNA
                                               from nucleases and genomic DNA from a foriegn bacteria in the
under its band, that looks like RNA.           culture. Alternatively, nucleases from bacteria that grow in the
                                               residual buffer in rig and tubing are breaking down DNA. Be
                                               certain that 2% bleach step and rinse step has been performed
                                               prior to the first run of the day and that 1X running buffer is made
                                               fresh before each run.




                                               7
                 PROBLEM                                                 SUGGESTED SOLUTION
3. Cams are stuck in down or other position and cassettes Press “interrupt” followed by the “A” key a few times to unstick the
will not fit into rig.                                    cams.Turn the instrument completely off, wait ten seconds, then turn
                                                          the instrument back on.

4. No bubbles appear at front electrode in rig during run.   Check all rig connections.
                                                             Alternatively, the running buffer may be made incorrectly and power
                                                             supply has shut off automatically. Press “Interrupt” followed by
                                                             “Drain”, then wait for buffer to drain. Turn off the instrument, wait
                                                             ten seconds, remake running buffer and start the program over.

5. Running buffer drains too slowly.                         Rig filter(s) are clogged with bits of agarose.      Remove rig filters
                                                             from the right corners of the rig with a plastic pipet tip or tweezers,
                                                             rinse with hot water in sink and replace.

6. Sample volume in microchamber block wells is greater A. Microchamber block has not drained. Slide the microchamber
than 35 ul at the end of run.                           block up before removing samples.
                                                        B. Running buffer is contaminated with dust fibers from paper tow-
                                                        els. Remove and clean rig filters, rinse rig with pure water prior to
                                                        beginning run. Always perform rinse at the end of the run.



7. Air bubble is trapped in sample well of                   Running buffer is contaminated with dust or fiber from paper towels.
microchamber block and no buffer volume is                   Rinse rig with pure water prior to beginning run. Always perform
obtained from sample well at the end of run.                 rinse at the end of the run. To prevent the problem entirely, pre-fill
                                                             microchamber block membrane chamber with 00 ul of running
                                                             buffer prior to beginning run. This step is easily accomplished with a
                                                             multi-channel pipettor. Use of 0.22 micron filtered water for making
                                                             running buffer will prevent problem.

8. Over Current or high current error message on display. Culture media loaded has too much salt (i.e > 0.5 gm/ liter). Termi-
                                                          nate run and regrow cultures in MR00 broth.

9. Instrument over fills with buffer.                        Float valve is dirty. Rinse valve with deionized water. Wipe off
                                                             float valve with a paper towel.

10. Purified DNA will not sequence on automatic DNA
                                                             Mostly likely too much template is being used. 1-2 ul of purified
sequencer.
                                                             DNA solution is recommended for concentrations ranging from
                                                             0.04 - 0.3 ug/ul of the final purified DNA.




For questions or problems please call us at -800-466-7949 or (858) 45-603, or send E-mail to macres@macconnell.com




                                                             8
I. Mini-Prep 96™ Automated Plasmid DNA Purification System Specifications
Instrument
         Weight uncrated:              5 lb. (.4 kg)
          Weight crated:               43 lb. (9.5 kg)
         Height: 9 in.       ( 3 cm.)
         Width:              4.5 in. ( 35.6 cm.)
         Depth:              8.5 in. (47 cm.)
         Work area recommended: 6 x 4 in. (4 x 6 cm.)
         (Instrument automatically fills with running buffer from a user-supplied reservoir at start of
         run and drains at termination of run.) The ABD-101 unit will automatically prepare the 1X
Running Buffer if installed with the instrument.
Operation Requirements
         98- 0 volts AC 50/60 Hz (USA, Canada and Japan)
         00 - 50 volts AC 50/60 Hz (Europe, Asia, and the Far East)
         Room Temperature Range Required: 18 - 25 ºC
         Buffer solution required: 000 ml for one run, or 000 ml per cycle.
Disposable Cassette        (C1202-SB only)
         Height:             0.9 in                      (.3 cm)
         Width:              4.6 in                      (.6 cm)
         Depth:              .375 in                    (7.0 cm)
         Weight:             70 gms                      (packaged 9 gms)
         Well capacity :  wells (.5 ml each for  ml C0-SB cassette, .0 ml for C40-SB cassette)
         Spacing of loading and sample wells: 0.355 in. (9 mm, for C00-SB cassette, 4.5 mm for
C40-SB cassette)
         Finished Sample Volume: 20 -25 ul, Separation material: modified agarose
Instrument Safety Features
         Automatic voltage shut-off when lid is lifted
         Non- conductive case
         Over-flow basin in case
         Power “on/off” switch located in rear instrument panel
Microprocessor-Controlled Electrophoresis System
         Voltage Range: 0 - 50 volt (direct current)
         Current Range: 0 - .6 amp
         Power Range : 0 - 40 watts
         Programmable Output - Voltage and time segments of run can be programmed by operator.
Automatic separation and electroelution are accomplished through use of the () computer controlled power supply,
(2) buffer filling and draining, (3) rotating cam activated microchamber elution block in disposable sample cassette.
Automatic bleach solution and Running Buffer preparation can be accomplished with the ABD-0 accessory.
Plasmid Purification Programs
         P.        70 min. plasmid high yield, C0-SB cassettes
         P.        60 min. plasmid high yield, C0-SB cassettes
         P3.        70 min. plasmid high yield, C1202-SB cassettes larger plasmid > 8 kb
         P4.        70 min. plasmid high yield, C40-SB cassettes, no buffer change
         G1.        90 min. genomic DNA
         U1.        Variable time, user programmable mode. Genomic or plasmid DNA
Sample Throughput, Plasmid
          to 96 samples per program. Example: 768 samples in eight to nine hours on program .
Rear Panel Fuse Specifications:
         Use only 1.25” X 0.25 “ cylindrical fuses. When viewing the instrument from the rear, the outer right hand
fuse should be a 50 volt 3 amp slow-blow fuse; the outer left-hand fuse should be a 50 volt, 5 amp slow-blow
fuse.
Warning: The instrument has been designed to protect the operator against electrical shock. However, to
be completely safe, wear latex gloves when inserting or removing cassettes from the rig. Do not insert metal
objects into the electrophoresis rig at any time. Do not negate lid switches.

                                                         9
          Appendix A: Mini-Prep 96 Flow Diagram of Run Steps and Display Screens
                                (Automatic Start Program)
�
                                      

������������������������������������MINI-PREP 96




















MACCONNELL RESEARCH CORP





AUTOMATIC BLEACH/BUFFER DILUTER DETECTED/ NOT 

DETECTED



ON BENCH, LOAD CASSETTES WITH CULTURES WITH COMBS 

AND PILLS. *PRESS START*





SET TIMER FOR LYSIS STEP?
YES 
NO

SET LYSIS TIMER FOR 5 MINUTES





PERFORM A RECOMMENDED BLEACH CYCLE? YES NO
             or  L of deionized water if ABD-0

                                                       detected


PLEASE PROVIDE 1L OF 2% BLEACH SOLUTION







PRESS 'START' TO STERILIZE THE SYSTEM





BLEACH CYCLE IN PROGRESS FILLING/DRAINING





PLEASE PROVIDE 1L OF DEIONIZED WATER AND PRESS
         or  L of pure water if ABD-0
  'START' TO RINSE THE RIG
                             detected




RINSE CYCLE IN PROGRESS 

10 SECONDS FOR RINSING *PLEASE WAIT*





PLACE CASSETTES INTO RIG, LOWER CAMS

  
WHEN FINISHED *PRESS START *





PLEASE PROVIDE 2L OF 1X BUFFER SOLUTION

RIG FILLS AUTOMATICALLY *PRESS START *







SELECT A RUN PROGRAM AND PRESS 'START'

P1    P2   P3    G1   U1



RUN       BACK

                                        Program automatically begins when ‘start” is pressed




PROGRAM WILL AUTOMATICALLY BEGIN IN 

______ MINUTES











BUFFER CYCLE IN PROGRESS





CHOICE IS: P1 PLASMID DNA *GENERAL USE* 70 MIN





RUN SUCCESSFULLY COMPLETED







                                                   0
Appendix B: User Programming Feature
             From program select menu

                  program U1                                Program U1: Programmable program. Each
                                                            program has two cycles: Separation and Elution .

                                                            Each cycle can have up to 4 steps. Within each step, 

                      Enter key                             there are choices for voltages and time. The

     STEPS IN THE SEPARATION CYCLE =               1        programming guide in Appendix B can help in the
                                                            planning of your program.
                                                            Choose the number of steps in the Separation cycle 

                      Enter key                             by pressing the arrow keys.
     ANY REVERSE VOLTAGE IN THE 1ST STEP ?
               YES           NO                             Each step has the option to have a forward and 

                                                            reverse pulse voltage. If "YES" is chosen, forward

                                                            and reverse voltage (50 to 150 volts) as well as

         Enter key                Enter key                 the corresponding time, need to be entered. The
    FORWARD VOLTAGE FOR THE 1ST SEPARATION                  reverse pulse time is in SECONDS (0 to 120
               STEP (volts) = 100                           seconds) and the forward pulse time is in MINUTES 

                                                            (0 to 120 minutes). The total time for the 

                                                            separation step should be more than the combined 

          Enter key                Enter key                pulse times.

        FORWARD PULSE TIME FOR THE 1ST                      

        SEPARATION STEP (minutes) = 0                       

                                                            If there are no reverse voltages in the separation 

                                                            step, the only information that needs to be entered

          Enter key                                         is the forward voltage and the total time for that

    REVERSE VOLTAGE FOR THE 1ST SEPARATION                  step. 

              STEP (volts) = 0                              

                                                            Press the arrow keys to scroll and the Enter key

                                                            to advance the menu.
          Enter key
        REVERSE PULSE TIME FOR THE 1ST
        SEPARATION STEP (seconds) = 0

          Enter key
    TOTAL TIME FOR THE 1ST SEPARATION STEP
               (minutes) = 30

                      Enter key

     STEPS IN THE ELUTION CYCLE =              1            The elution cycle also has 4 possible steps, but

                                                            has no reverse pulse voltage option.

                      Enter key
    FORWARD VOLTAGE FOR THE 1ST ELUTION
               STEP (volts) = 100

                      Enter key
    TOTAL TIME FOR THE 1ST ELUTION STEP
                (minutes) = 30

                      Enter key
      PROGRAM 6        TOTAL TIME: XXX MINUTES              Program introduction screen will show the 

                                                            total program minutes.




                                                       
Appendix B. Programming Screens (continued)




                        After the

                  program introduction

                     (programs U1)
                                                       Minutes remaining screen will be updated

    MINUTES REMAINING IN THIS RUN: XXX                 every 30 seconds. After the separation steps,

                                                       the program would either change the buffer

                                                       and/or push the micro chamber down before

                                                       the elution cycle.

             DRAIN CYCLE IN PROGRESS                   The drain cycle is timed for approximately 1
                                                       minute.


       PLEASE REMOVE CASSETTE AND SAMPLES
                                                       The program will wait until the lid is open before

                                                       showing the start screen.

       PLEASE PROVIDE 1L OF DEIONIZED WATER

        AND PRESS 'START' TO RINSE THE RIG

          fill cycle followed 

          by drain cycle
            

            to the LOAD SAMPLE CASSETTES screen        At almost any time during the program, the 

                                                       Interrupt Key can be pushed to stop the program.

                                                       By pressing the Start Key , the program will return

                  INTERRUPT SCREEN                     to the start screen. The Continue Key will resume

                                                       the program, but not necessarily where it was
    'ENTER' = RESTART, 'CONTINUE' = RESUME             stopped.

    'DRAIN' = DRAIN, 'A' = Cam shaft rotate            The Drain Key will drain the electrophorisis rig 

                                                       and then start the program over. The "A" key 

                                                       will rotate the cam shafts, then return to

                                                       the interrupt screen.
                     ERROR SCREENS

                                                       The lid cannot be open during the Fill Cycle and 

               THE LID IS OPEN

                                                       during the run. All functions are suspended until

       PLEASE CLOSE THE COVER TO CONTINUE
                                                       the lid is closed.



     VOLTAGE ON   PLEASE REFER TO THE MANUAL
          MP-24 or 96 machine has detected voltages across 

         AND PRESS 'CONTINUE' TO RESUME                the electrodes when no voltages are applied. This is

                                                       a hazardous condition and MacConnell Research

                                                       should be contacted immediately.

                                                       If there is not enough buffer in the resevoir for the 

        PLEASE ADD BUFFER TO THE RESEVOIR

                                                       pump to fill up the rig in a certain time, the
         AND PRESS 'CONTINUE' TO RESUME
                                                       MP-24/96 will pause and ask the user to add
                                                       buffer to theresevoir before continuing.

                                                       The MP-24/96 machine has detected that the
    OVER CURRENT!PLEASE REFER TO THE MANUAL
           amperage draw from the rig is beyond the expected
        AND PRESS 'CONTINUE' TO RESUME                 norm. The most likely situation is that the buffer is
                                                       made too strong.




                                                  
Appendix B (continued): User Programming Feature

        To Program the Mini-Prep 96™ using a custom voltage program, select the “U” option from the program
menu. Lightly press the “Start/Enter” key to program the separation and electroelution run stages. The hierarchy of
terminology being used in this explanation is that a run program contains “stages”, which contain “steps.”

General guidelines are as follows:
          There are two main stages to the run, the separation stage and the electroelution stage. The user-program-
mable software will automatically activate the rotating cam shafts to push down the microchamber block prior to the
electroelution stage of any custom program. In addition, the instrument will automatically drain the rig at the end
of the eletroelution stage(s). Therefore, you do not have to program these functions. A diagram of the Program U
menu is depicted in Appendix A, along with a programming guide in Appendix B.
          Once program U is selected and the “Start/Enter” button has been pressed, several menu options will be
presented. The following steps detail how to create your own custom program.

Step . Select the number of steps in the separation stage as , , 3 or 4. These represent periods of run time at a
given programmed voltage during the separation stage. Increase the number of steps by pressing the “right” arrow
on the key pad or decrease the number of steps by pressing the “left” arrow on the key pad. Once you have selected
a number, press “Start/Enter”.

Step 2. Program a time for the first separation step by using the right and left arrow keys to set the desired time,
displayed as a number, at the end of the step descriptor phrase. Press “Start/Enter” once to lock in that time. Then
program the voltage by using the right and left arrow keys to set the desired voltage. Press “Start/Enter” to lock in
that voltage. A new menu for the second step of the separation stage will appear. Repeat the above process for step
two of the separation stage and subsequent separation steps until the voltage and time for all steps of the separation
stage have been programmed. If you happen to press the “Start/Enter” key more than once, the software will skip a
step. If this occurs, you can begin the programming function over again by pressing “Interrupt”, then “Start/Enter”
to go to the beginning step of the programming process.
          Note: The total number of volt-minutes (volt x minutes) for the separation phase should add up to or
equal 000 for plasmids greater than  kb. As an example, you might have selected two steps for the separation
stage : step , 50 volts for 8 minutes (400 volt-minutes), and step , 00 volts for 6 minutes (600 volt-minutes) for
total volt-minutes of 000.

         Low voltage at the beginning releases more plasmid from the lysis mixture by allowing smaller molecules
to move out of the mixture prior to separation. Most of the yield loss comes from the lack of plasmid movement out
of the well even after the run has been completed. Slow starting (50 volts for 5-10 minutes), moves more DNA into
the gel and allows it to focus prior to entering the gel.

Step 3. The electroelution stage menu will appear after the last entry in the separation stage has been programmed.
The electroelution stage is programmed the same way as above with up to four steps. Do not exceed 50 volts on
the electroelution or automatic shut-off of the power supply will occur. Here, the optimal voltage happens to be 00
volts. Thirty minutes at 00 volts is minimal for collecting plasmids -7 kb. Longer electroelution times will collect
bigger plasmids and give higher yield. You can lower the voltage and electroelute for longer time, if desired. The
quality of the DNA is rarely effected by the electroelution stage.
          For more details and guidelines on programming the instrument, call MacConnell Research Corporation at
-800-466-7949




                                                          3
Appendix C: Procedure for Lysis and Loading Samples with Lysis Buffer A and B (Pre-Mixing)

          . Thaw one vial of Lysis Buffer A per cassette and the Lysis Buffer B vial.
          . To prepare a sample for one lane of the cassette, pipet 0.9 to .0 ml of freshly grown E. coli culture
containing plasmid DNA into a disposable tube, add 100 ul of Lysis Buffer A and mix thoroughly. Incubate this so-
lution for 4-5 minutes at room temperature. Incubation for up to 0 minutes may be necessary to complete the lysis.
        Important note: The lysis mixture should become viscous at this point. If the mixture does not become vis-
cous, as can occur with some strains, freeze the mixture to solid and then thaw before going to step 3. Freezing can
be accomplished rapidly in dry ice or in an ordinary freezer. Bacterial culture can be stored frozen prior to
addition of Lysis Buffer A to give the same effect. Freezing does not decrease the quality of the DNA obtained.
Higher yields can be obtained by spinning down 2-5 ml of bacteria, then resuspending in 0.5 ml of 1X Running Buf-
fer prior to addition of 00 ul of Lysis Buffer A and B.
          3. Add 0 ul of Lysis Buffer B to each sample tube from above. Mix thoroughly and allow this solution to
stand while preparing the cassette and microchamber block. Once Lysis Buffer B has been added, the solution is
stable for hours.
          4. Load the lysed samples from step 3 into separate wells of the cassette. It is possible to load up to .0 ml
of lysed culture into one well (0.9 ml of bacterial culture + 0.12 ml of Lysis Buffers.)

Procedure for Mixing Lysis Buffer with Culture Without an Intermediate Tube

         The lysis buffers and bacterial culture can be mixed in the sample well directly by the following procedure.
         () Pipet 00 ul volume Lysis Buffer A into a cassette well.
         () Pipet 0.5 to 0.9 ml of bacterial culture into the well with above buffers and mix by pipetting up and
down. This is best accomplished with the pipet tip raised above the bottom of the sample well floor to prevent the
thin agarose medium comprising the well floor from clogging the pipet.
         (3) Pipet 0 ul of Lysis Buffer B into well and repeat the above mixing step. Activate program.

Appendix D Midi-Preps (Mid-Scale Preps) on the Mini-Prep Instrument.
        To perform larger scale preps on the Mini-Prep 4 or 96, simply load multiple lanes of a cassette
with the same culture. Culture volumes greater than 2 ml volume should be grown in an erlenmeyer flask
for 18-20 hours. The purified DNA from multiple lanes can be concentrated by ethanol precipitation.
Once this is carried out, the concentration of the resulting DNA can be determined by OD260 reading.

Appendix E. PCR Band purification on the Mini-Prep 96

        PCR amplified bands can be easily purified from unreacted primers, nucleotides, and enzymes
using the Mini-Prep instrument. Follow the procedure below.
        1. Diluted the PCR reaction to 100-150- ul with 1X Running Buffer
        . Prepare a -well cassette by raising it microchamber block
        3. Load the diluted PCR reaction mix into one lane of the cassette. One reaction per well.
        4. Run the instrument as you normally would (bleach, rinse and prepare x Running Buffer).
                Choose the User Program U from the menu and program the instrument for:
                        a. 5 minutes separation at 00 volts followed by a buffer change
                        b. 45 minutes electroelution at 00 volts.
        5. When the instrument drains, remove the purified PCR fragment from the well(s)




                                                        4
Appendix F. Genomic DNA Purification
Technical Brief
Genomic DNA User Protocol for the MP96 (Mouse Tail Snip)
This protocol was used for the purification of mammalian genomic DNA from mouse tail snips. This same
protocol can also be applied to genomic DNA isolation from tissue culture cells, dissected tissue and bacterial cells.
Steps:
. 0.5 – 0.75 mg of tissue is placed in a .5 mL eppendorf tube.
. Add 00-500 ul of the digestion solution to the eppendorf tube and incubate at 55 C for  hours to overnight (until
    tissue is completely digested).
         Digestion Solution:
                   0.1% SDS
                   50 mM Tris pH 7.5
                   0 mM EDTA
                   0.35 mg/mL Proteinase K
3. Centrifuge @ 3000 rpm for 5-0 min (this step may not be needed)
4. Remove supernatant and put in a clean .5 mL eppendorf tube.
5. 50 and 00 ul of the resulting supernatant was brought up to 500ul and loaded into the sample wells of a  well
    cassette on the MP96 instrument. The microchamber block of the cassette was removed and discarded.
    Be sure the rig in the MP96 instrument is bleached and rinsed.
6. Prepare 2 Liters of 1X Running Buffer for this program.
PROGRAM: Choose U for user input. The program is as follows:
                   30 min forward @ 00V
                   5 min reverse @ 00V
                   30 min forward @ 00V
                   5 min reverse @ 00V
                   Buffer Change
                   30 minutes forward @ 00V
                   5 minutes reverse @ 00V
                   End Program
7. After the program has ended and the rig has fully drained, collect all of the sample directly in the sample loading
wells. In some cases, you may discover that the final volume is slightly higher than your starting volume. This is
normal.
         3-0 ul of the collected sample can be used directly in a 5 ul PCR reaction, or the entire same can be ethanol
precipitated to reduce the genomic DNA sample volume.
         Example of PCR reaction with Tail Snip genomic DNA purified by the above procedure.
         Figure 1. PCR of Tail Snip Genomic DNA
                                               HSP set 1          HSP set 2        HSP set 3




Lanes in the above gel contain the PCR reaction from amplification of 7 ul (of 750 total recovered) of automatically
purified mouse tail genomic DNA. Primer sets were designed to match mouse heat shock genes (HSP sets 1, 2, and 3).
PCR reaction was performed on a MJ Research thermocycler for 30 cyles.

                                                          5
Note: For other types of genomic DNA contact MacConnell Research Corp. 800-466-79049 macres@macconnell.com
                         Mac Connell Research Price List

Catalog Number Item Description                                                     Price

C0-SB                -Well Sample Cassette, with applicator combs               $5
                        and Lysis Tablets 0 pack, (0 samples)
C40-SB                4-well Sample cassette, with lysis buffers
                                 8 pack, (9 samples),                              $7
C1500                   100X Running Buffer Stock for MP-24 and 48, 250 ml           $18
FSP96                   De-Salting plate for fluorescent DNA sequencing              $19
MR00-3                Enhanced Growth Media for Plasmids (300g)                    $46
MR00-0               Enhanced Growth Media for Plasmids ( kg)                    $49
MR00-0               Enhanced Growth Media for Plasmids ( kg)                    $85
ABD-0                 Automatic Buffer Diluter                                     $695
MG-384                  Mini-Grow 384 Shaker                                         $3700
MPT-00                 Microporous tape, for sealing deep-weel blocks during growth $8
DWB-96                  Deep Well Block (96 Well) for Bacterial growth (0 pack)     $6




                                          6
                              CERTIFICATE OF ANALYSIS

                                                MP-9600
                          Instrument Model ________________________

                          Serial Number:    _________________________




                         Date Tested:           _______________________

                         Test Performed By:   _________________________

                         Yield Data:

                          Plasmid DNA yields from multiple 2.0 ml E. coli cultures containing
All MacConnell            pBluescript (pMAC-1) grown in MR2001 media for 20 hours at 37ºC.
Research
products are tested
as needed to ensure
given performance
specifications.




695 Cornerstone Court
San Diego, CA 9
858-45-603
Fax: 858-45-6753
-800-466-7949
                         Certified By: ______________________________________________


                                                7
MINI-PREP 96™
  New Model

 Instruction Manual




  6195 Cornerstone Court E.
    San Diego, CA 92121
       1-800-466-7949
        858-452-2603

E-mail macres@macconnell.com
     www.macconnell.com




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