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					                                                     Ana M. Aguirre-Arteta, August 1998



A     RNA PREPARATION AND NORTHERN HYBRIDIZATION



GENERAL:


* Always wear gloves while handling reagents and RNA samples to prevent RNase
contamination.


* Change gloves frequently and keep tubes closed.


* Work quickly and keep everything on ice when possible to avoid degradation of RNA by
endogenous or residual RNases.


* Throughout the protocol, deionized water (MilliQ grade) was autoclaved twice and used
for all solutions except for preparation of the probe. For the latter, DEPC-treated water
should be used (0.1% DEPC in deionized water allow standing overnight and then
autoclave).




DISPOSABLE PLASTICWARE


* The use of sterile, disposable polypropylene tubes is recommended. These tubes are
generally RNase-free and do not require pretreatment to inactivate RNases.



NON-DISPOSABLE PLASTICWARE


* This should be treated before use by rinsing it with 0.1M NaOH, 1mM EDTA followed by
a rinse with water.



GLASSWARE


3.    This should be cleaned with 0.5%SDS, then rinsed with water, autoclaved and
      oven baked at 180oC for four hours or overnight.
                                         1
ELECTROPHORESIS TANKS


* They should be cleaned with 0.5%SDS and rinsed with water, and then rinse them with
pure ethanol and allowed them to dry.



STORAGE OF RNA


Total RNA may be stored at -70oC (or lower temperature) in water.


Alternatively after Qiagen RNA extraction and after eluting the RNA from the columns with
water, either dry down the sample, or precipitate with 3 M Sodium Acetate and 100%
ETOH. Then resuspend in formamide.


Resuspending RNA in Formamide (Chomczynsky, P. Solubilization in Formamide protects
RNA from degradation. Nucleic Acid Res. 20 (14) : 3791-3792) will protect RNA from
RNases. The RNA is then very stable, allowing it to be stored overnight at 4 oC, or
indefinitely at -20oC. Samples can also be left temporarily at room temperature without
degradation.



B     EXTRACTION OF RNA


Use the Qiagen RNeasy Midi Kit\50\Cat Number 75144 kit and follow the instructions in
the respective manual.
Total RNA extractions are done at room temperature and can also be done with frozen
cell pellets. When cytoplasmic RNA is extracted the cell pellets used should be fresh and
the RNA is extracted keeping all the reagents at 4 oC. The minimum amount of cells that
can be used are 5 x 106 and the maximal 1 x 108 for the Midi-Kit.



C     RNA QUANTITATION



The concentration and purity of RNA can be determined by measuring the absorbance at
260 nm and 280 nm in a spectrophotometer. RNA concentration is determined by making
a 1:50 dilution in water. An absorbance of 1 unit at 260 nm corresponds to 40 g of RNA
per ml.


                                         2
An example of the calculation involved in RNA quantification is:

A260 = 1 = 40g/ml (in water)
Volume of RNA sample = 450 l
Dilution = 10 l of RNA sample + 490 l dH20 (1:50 dilution)
Measure dilution A260 = 0.55
Concentration of RNA stock = 40 x A260 x dilution factor = 40 x 0.55 x 50 = 1100 g/ml
Total yield = concentration x volume of stock in ml = 1100 g/ml x 0.45 ml = 495 g

The ratio between the readings at 260 nm and 280 nm (A260/A280) provides an estimate
of the purity of RNA. Pure RNA has an A 260/A280 ratio of 1.8-2.1 in 10 mM Tris-HCl pH
7.5.




D      SAMPLE PREPARATION/CONCENTRATION



D.1    SAMPLE PREPARATION


To 4 l of sample (corresponding to 30g RNA) add 10l sample buffer and heat it 10
minutes at 55oC.
Add 4 l dye solution. Heat the mixture at 65oC for 10 minutes, put onto ice, quick spin
and load onto the gel and wait a few minutes until the samples are into the well.

* Sample buffer:
5 x running buffer                 100 l
formamide                          1 ml
(37%) formaldehyde                  356l
(It can be aliquotted and kept at -20oC)


* Dye solution:
7.5% ficoll 400 (Pharmacia)        750 l
0.1% BPB                           0.01 g
dd H2O                             9.25 ml
(Keep at 4oC)




                                             3
D.2   SAMPLE CONCENTRATION


When the volume of the sample exceeds 4l it is necessary to concentrate the sample
by;
* Ethanol precipitation:
- Add 1/10 volumes of 3 M sodium acetate pH 5.2 to the sample. Mix.
- Add 2.5 volumes of 100% ice-cold ethanol. Mix and leave it at -86oC for 1/2 hour
- Spin 15 minutes at max. speed in the cold, pour off supernatant
- Wash with 70% ice-cold ethanol and spin again at max. speed for another 15 minutes in
the cold, pour off supernatant
- Vacuum briefly to dry the pellet and dissolve it in 4l of water.


The RNA samples could also be quantified and the quality evaluated by running a 1%
agarose gel with 1 l RNA (=0.5 g/l)




                   Fig 1.Total RNA from MEL, C2C12 MB
                   and C2C12 MT (lanes 2, 3 and 4
                   respectively) on a 1% agarose gel.
                    Lane 1 is a 200 bp Smart-Ladder/
                          Eurogentec




                                             4
E     RNA GEL ELECTROPHORESIS



E.1 GEL (Agarose FMC. Nu-Sieve Agarose)
Weight 0.6 g agarose and dissolve in:


                                  (ml)           Final concentration
5x formaldehyde buffer            10            1x
dd H2O                            31.1
dissolve the mixture in a microwave and cool it down, then add:
(37%) 12.3 M Formaldehyde         8.9           2.2 M



E.2   5x FORMALDEHYDE GEL RUNNING BUFFER:
                                   Final concentration
MOPS (pH 7)              20.6 g    0.1 M
Sodium acetate           3.28 g    40 mM
0.5 M EDTA (pH 8)        10 ml     5 mM


Dissolve 20.6 g of 3-(N-morpholino) propanesulfonic acid (MOPS) in 80 ml of 50 mM
sodium acetate. Adjust the pH to 7 with 2N NaOH. Add 10 ml of 0.5 M EDTA (pH 8).
Adjust the volume of the solution to 1 liter with deionized water.


Sterilize the solution by filtration through a 0.2-micron Millipore filter. Store it at room
temperature protected from light.



E.3 ELECTROPHORESIS OF RNA
Before loading the samples onto the gel, run the gel for 5 minutes at 85 V, then run it with
the samples at 85 V (for small gels).


The gels can be run at 60 V for 3 hours for the small gels or overnight: 15 V for the small
gels and 30 V for the big gels.




                                           5
E.4   STAINING OF THE GEL
The gel is stained with Sybr Green II (5 µl/20 ml 0.1x TBE buffer) for 1/2 hour. The gel can
then be visualized with a transiluminator and also with a Fluorimager.




                           Fig. 2: Same samples as in Fig.1. but this
                           time run in a 1.5% formaldehyde gel, 30 g
                           of total RNA were run together with 6 l RNA
                            marker (NE Biolabs). The RNA marker was
                           run with 1 l of Ethidium Bromide (1mg/ml).




F     RNA GEL BLOTTING



F.1   Capillary transfer


Soak the gel for 20 minutes in 0.05 N NaOH. This partially hydrolyzes the RNA and
improves the efficiency of transfer. Then rinse the gel in deionized water and soak it for 45
minutes in 20x SSC.


1.    Transfer the gel to a dish and trim away any unused areas of the gel with a razor
      blade.


2.    Place a piece of Whatman 3MM paper on a glass plate to form a support that is
      longer and wider than the gel. Place the support inside a large dish. Fill the dish


                                             6
      with 20 X SSC until the level of the liquid reaches almost to the top of the support.
      When the 3MM paper on the top of the support is wet, get rid off air bubbles with a
      pipette rod.


3.    Cut a piece of nylon membrane about 1 mm larger than the gel in both dimensions.
      Use gloves to handle the membrane.


4.    Wet the membrane with deionized water and then immerse it in 20 X SSC for at
      least 5 minutes.


5.    Place the gel on the support in an inverted position so that it is centered on the wet
      3MM paper. Make sure that there are no air bubbles between the 3MM paper
      and the gel.


6.    Surround the gel with Parafilm. This serves as a barrier to prevent liquid from
      flowing directly from the reservoir to paper towels placed on the top of the gel.


7.    Place the filter on top of the gel so that the corners are aligned.


8.    Wet two pieces of 3MM paper (cut to the same size as the gel) in 20 X SSC and
      place them on top of the wet filter. Get rid of bubbles as mentioned before.


9.    Cut a stack of paper towels just smaller than the 3MM papers. Place the towels on
      the 3MM papers. Put a glass plate on top of the stack and press it down with a
      weight.


10.   Allow transfer of RNA to proceed overnight. As the paper towels become wet, they
      should be replaced.




                                           7
             Fig .3: Picture showing capillary transfer set up.




11    After transfer is completed, the membranes should be rinsed briefly in 2 X SSC,
      0.1% SDS and allowed to dry at room temperature


12.   To fix the RNA to the membrane place the dried membrane between two pieces of
      3MM paper and bake for 2 hours at 80 oC in an oven. Optional; the membrane
      can also be crosslinked at 120 millijoules for a few seconds.



F.2   Semi-Dry transfer


For semi dry Transfer, follow the protocol from the manufactures (Hoeffer).
If the gel contains formaldehyde, remove it by soaking the gel in 0.1 X TBE buffer for 1.5
hours at room temperature. This will also lower the ionic strength of the buffer in the gel
from 1 X to 0.1 X. The nylon membrane should also be soaked in water for 5 minutes and
then in 0.1 X TBE buffer also a few minutes.


Gel Stack Set up
Cut 10 sheets of blotter paper (Gel Blot Paper/GB002-33930/Schleicher & Schuell) and
one piece of nylon positively or not charged membrane (Amersham) to the size of the gel.
The positively charged membrane is not recommended if it has to be reprobed several
times because of higher background levels but the sensitivity is higher. The nylon

                                                 8
membranes are more resistant than the nitrocellulose ones. Do not cut the paper or
membrane larger than the gel. If the papers from either side of the stack touch each other
during the transfer, the current will travel throughout the paper, not through the gel.


Saturate the blotter paper in 0.1 X TBE buffer. Soak the membrane in distilled water for 5-
10 minutes, then in 0.1 X TBE buffer for 20 minutes. (A nylon membrane must be used in
this procedure because unlike nitrocellulose, nylon membranes will retain RNA in low ionic
strength buffers).


Use a pre-cut Mylar mask, and cut a hole about 2 mm smaller on each side than the size
of the gel.


Stack as follows; (between each layer, roll a pipette over the stack to remove any trapped
air bubbles)
* Place the Mylar sheet on the bottom of the unit (anode)
* Stack 5 sheets of wet blotter paper over the hole in the Mylar sheet
* Place the presoaked nylon membrane on top of the blotter paper
* Place the gel on top of the membrane
* Place 5 sheets of wet blotter paper over the gel
* Put the lid (cathode) over the whole stack


Transfer at 50 mAMP (6-7 V) for 2 hours.
Remove the membrane and wash it with 6 X SSC for 10 minutes

Bake for 2 hours at 80oC or cross-link at 120 milijoules for a few seconds between
Whatmann paper. Once the filter is baked, it can be stored at room temperature wrapped
in aluminium foil for weeks. If the filter is stored, it is important to rebake it before
prehybridization (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A.,
Seidman, J. G., Struhl, K. Current Protocols in Molecular Biology, pp. 4.9.8. 1988).



Reagents:


5 X TBE Stock:      54 g Tris base
                    27.5 g boric acid
                    20 ml 0.5 M EDTA (pH=8)




                                           9
20 X SSC Stock      Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of
                    water. Adjust the pH to 7 with a few drops of a 10 N solution of NaOH.
                    Adjust the volume to 1 l with water. Sterilize by autoclaving.


G     LABELING OF PROBES


A simple method to transcribe cloned segments of DNA in vitro into highly radioactive
single-stranded RNA is shown in Fig 3. These transcripts can be used as probes and they
can be separated from the DNA template without gel electrophoresis.


Advantages of RNA probes:


* They form hybrids with both DNA and RNA that are more stable than DNA:DNA hybrids
(Casey J and Davidson N, Rates of formation and thermal stabilities of RNA:DNA and
DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 4:
1539,1977).


* Both DNA: RNA and RNA:RNA hybrids are resistant to digestion with RNAase, and it is
therefore possible to use this enzyme to remove any probe that is nonspecifically bound
without affecting the DNA:RNA or RNA:RNA hybrids (Sambrook, J., Fritsch, E. F. and
Maniates, T. Molecular Cloning. A Laboratory Manual. pp. 10.9. 1989).



G.1   RADIOACTIVE PROBES

A     RIBOPROBE PREPARATION


Transcription of RNA from the plasmid of interest will contain at least one of the following
RNA polymerases: T7, SP6 or T3. Because these polymerases are extremely promoter-
specific, virtually homogenous RNA can be obtained using plasmid DNA as the template
in a transcription reaction.


When it is desirable to copy only insert DNA sequences, the plasmid is linearized at an
appropriate restriction site prior to the transcription reaction and only discrete runoff
transcripts are obtained, virtually free of vector sequences.


RNA transcripts can be used to generate probes for hybridization to Northern blots.



                                          10
Hybridization with RNA probes allows the background on blots to be further reduced by
RNase A treatment to remove nonspecifically bound probes, a step that is not possible
with DNA probes.


RNase A treatment ('Protocols and Applications Guide', 2 nd Edition. Promega, page 66):
After hybridization, rinse the membrane 3 times, 5 minutes each in 2 X SSC and then
incubate it at room temperature for 15 minutes in 2x SSC containing 1 mg/ml RNase A.
Rinse the filter for 30 minutes at 50oC in 0.1 X SSC + 0.1% SDS




                   Fig. 4: Diagram showing Riboprobe labeling




      DNA template preparation


      Transcription of RNA from the desired plasmid is performed with SP6, T7 and T3
      polymerases

      Important considerations;
                                           11
*    The plasmid has to be completely linearised, because a small amount of
undigested plasmid DNA can give very long transcripts which may incorporate a
substantial fraction of the radiolabeled rNTP.
* Enzymes that produce 3'end protruding ends should not be used (e.g. ApaI, PstI).
These enzymes can give strange transcripts than can contain sequences
complementary to the expected transcript as well as sequences corresponding to
vector DNA. If there is no alternative restriction site, the 3' overhangs can be
converted to a blunt end using the 3'-5' exonuclease activity of Klenow DNA
polymerase.


Method;
1.   Restriction of the plasmid
             1.1.   An amount of 2 g of plasmid is restricted in a total volume of
                    20 l
             1.2.   An aliquot of 1l out of 20l is run on a 1% agarose to check
                    whether is linearized.


2.     Purify the restricted fragment by Phenol/Chloroform/Isoamyl alcohol
       extraction (Current Protocols in Molecular Biology, page 2.1.1) or by
       Phenol/ Chloroform (Maniatis, page 458). Here the first approach is used;
               2.1   First adjust the volume to 100 l in a 1.5 ml eppendorf tube, it
                     is important not to reduce this volume because sometimes the
                    presence of high salt concentrations can cause the inversion
                    of the aqueous and organic phases.
             2.2.   Add an equal volume of phenol/chloroform/isoamyl alcohol
                    (25/24/1)
             2.3.   Vortex strongly for 1 minute
             2.4.   Spin 15 seconds at room temperature in a microfuge (13000
                    rpm). Phases should be well separated, if not spin longer.
             2.5.   Remove the top aqueous phase containing the DNA and
                    transfer it to a new 1.5 ml eppendorf tube.
             2.6.   Optional: recovery can be improved by reextracting the
                    organic phase.


3.     Proceed with an Ethanol precipitation (Promega method from the
       Riboprobe leaflet);
             3.1. In a 1.5 ml eppendorf tube add 1/10 volumes of 3 M
                    sodium acetate pH 5.2, mix by vortexing.


                                   12
             3.2.  Add 2.5 volumes of ice cold 100% ethanol, mix by vortexing
             and place it at 86oC for 1/2 hour
             3.3.   Spin for 15 minutes at 13000 rpm in the cold. Pour off
                    supernatant.
             3.4. Wash with 1 ml 70% ethanol, mix and spin again as above.
             3.5. Dry off briefly the pellet in a Speed Vac, and dissolve in 6 l
             of water
             3.6. Run 1l out of 6l on a 1% agarose gel together with 0.5 g
                    DNA molecular weight marker, to determine the amount of
                    purified probe by comparing it with the marker.



Labeling of the probe

Use the Riboprobe kit from Promega to produce a RNA probe.
                                                                  (l)
              5X transcription buffer                             4
             100 mM DTT                                           2
              (40 U/l) RNasin                                    0.5
             ATP, GTP, UTP, dd H2O (2.5 mM each)                  4 (1l each)
             100 mM CTP                                           2.4
             DNA template                                         1 g
             32P CTP (50 Ci, at 20 Ci/l, Amersham)             2.5
             RNA polymerase (SP6, T7 or T3) (at 15-20 U/l)       1
             up to 20 l with dd H2O
Incubate the mixture at 37oC for 2 hours.


Important considerations;
* All the reagents must be at room temperature
* Add the reagents in the order indicated
* Warm the mixture briefly before adding the template at 37 oC
(Note: it is important that the template is added to previously warmed reagents to
avoid precipitation of the DNA due to the spermidine present in the transcription
buffer, see Nucleic Acids Research, Vol. 12, No 18, page 7035-7056,1984).




                                 13
B     RANDOM PRIMER LABELING
The procedure relies on the ability of random hexanucleotides to anneal to multiple sites
along the length of a DNA template. The enzyme synthesizes new DNA by incorporating
nucleotide monophosphates at the free 3'-OH group provided by the primer. The newly
synthesized DNA is made radioactive by substituting a radiolabeled nucleotide for a
nonradioactive one in the reaction mixture.
Use the Amersham MegaprimeTm DNA labeling kit.



      DNA TEMPLATE PREPARATION


      Digest the plasmid of interest with the appropriate enzymes to get the insert out.
      See above for running of the gel and purification.



      Labeling of the probe



      1.     Add the following components to a clean microcentrifuge tube;
                                                           (l)
             DNA (25 ng)                                   4
             dd H2O                                        12
             Random oligonucleotide primers                 5


      2.    Heat reaction tubes in a boiling water bath for 5 minutes and then centrifuge
            briefly at room temperature. If the DNA sample is in low melting temperature
            agarose, after removal from the boiling water bath and centrifugation, place
            the reaction tube at 37oC and then proceed to step 3.
            If the DNA sample is in aqueous solution, leave the reaction tube at room
      temperature and then proceed to step 3.


      3.     Add the following reagents to the reaction tubes:
                                                      (l)
             Unlabeled dNTPs                          4 from each except the
                                                       labeled one
             5 X reaction buffer                      5
             (10 X buffer containing Tris/HCl pH 7.5, 2-mercaptoethanol and MgCl2)
             Labeled nucleotide (dATP+dCTP)           5 l or 50 Ci each nucleotide
             (3000 Ci/mmol, -32PdATP+dCTP)
                                         14
            Klenow enzyme (5U/l)                         2
            Mix the contents with a pipette.


      4.    Incubate the reaction at 37oC for 1 hour.


      5.    After the incubation period, stop the reaction by adding 5 l of 0.2 M EDTA,
            pH 8.




            Fig .5: Diagram showing Random-primer labeling.




I     REMOVAL OF THE DNA TEMPLATE FOLLOWING TRANSCRIPTION
Add RQ1 RNase free DNase to a concentration of 1U/g of template DNA. Incubate at
37oC for 15 minutes, this is done immediately at the end of the labeling.



H     NON-RADIOACTIVE PROBES
The Riboprobe kit from Boehringer was used to produce a RNA probe
                                                       (l)
                    Purified template (25ng/l) 4 (1 g)
                    NTP (DIG-UTP)                      2
                    Buffer                             2

                                           15
                    RNase inhibitor (20U/l)             1
                    RNA polymerase (20U/l)              2
                    dd H2O up to 18 l
Incubate the mixture at 37oC for 2 hours
Add DNase I-RNase free (2 l) and incubate at 37oC for 15 minutes
Add 2 l of EDTA pH 8.


Precipitate the reaction by adding;      2.5l of 4M LiCl
                                         75 l prechilled 100% ethanol
Leave at -86oC for 1/2 hour
Spin at 13000 g for 15 minutes in the cold
Discard supernatant
Wash with 500 l of cold 70% ethanol
Dry briefly under Speed Vac. and dissolve pellet in 100 l of water


Use 0.2 g of probe/ml of hybridisation buffer


Detection (Boehringer Kit)
(1x) Wash buffer                                             2 minutes
(1x) Block buffer made in buffer 1= Maleic acid buffer       30 minutes
Antibody diluted in buffer 2 = block. buffer (75mU/ml)       30 minutes
(1x) Wash buffer                                             2 X 15 minutes
(1x) Equilibrate with buffer 3                               2 minutes
CSPD (1:100 in buffer 3), this can be reused                 5 minutes
Incubate the membrane at 37oC                                10 minutes
Film exposure for 10 minutes-1 hour




                                          16
     Fig. 6: Diagram showing DIG-Riboprobe labeling and different ways of detecting the bound nucleic
     acid with the anti-DIG-antibody.




I    DETERMINATION OF PERCENT INCORPORATION AND PROBE SPECIFIC
         ACTIVITY. TCA PRECIPITATION



1.   If 50 Ci was used, the dpm used is (50 x 2.2 x 10 6 dpm/Ci) = 110 x 106 dpm in
     a total of 20 l, so 5.5 x 106 dpm/l

2.   Determine the percent of incorporation using TCA precipitation. First, make a
     dilution of 1:10 of the labeled probe in water. Spot 1l of the 1:10 dilution onto
                                            17
      duplicate glass fiber filters (Whatman GF/A) and let this air dry. Count these
      directly to determine the total cpms.


3.    In duplicate tubes, add 1l of the 1:10 dilution to 100 g of fish sperm DNA in a
      total volume of 100l, mix and than add 0.5 ml of ice cold 5% TCA and mix again.
      Leave on ice for 5 minutes.


4.    Apply the samples to wet (with 5% TCA) filters under vacuum and wash twice with
      5 ml of ice cold 5% TCA. Rinse the filters with 2 ml of acetone and let them air dry.


5.    Place the dry filters into scintillation vials and add 4 ml scintillation fluid and count
      the samples.
      TCA precipitated cpm/ total cpm x 100 = percent incorporation


6.    If 1l of a 1:10 dilution was TCA precipitated, 10 X cpm precipitated = cpm/l
      incorporated. In a 20 l reaction, the total cpm incorporated is 20 times this
      number.


If 50 Ci of labeled CTP at 800 Ci/nmol were used, then 0.0625 nmol of CTP were
added to the reaction. If 2.4 l of cold 100 mM CTP were added to the reaction, then an
additional 0.24 nmol of CTP are included in the reaction, resulting in a total of 0.3025 nmol
of CTP.


If there was 100% incorporation and CTP represents 25% of the nucleotides in the probe,
then 4 X 0.3025 = 1.21nmol of nucleotides were incorporated and 1.21 X 330 ng/nmol =
399.3 ng of RNA were synthesized. Then % incorporation X 399.3 ng = total ng RNA
made.



J     REMOVAL OF UNINCORPORATED NUCLEOTIDES



Unincorporated nucleotides are removed from the labeled probe by size exclusion
chromatography through a Sephadex G-25 column (see table 1).


The column was made as follows (Sambrook, J., Fritsch, E. F. and Maniates, T.
Molecular Cloning. A Laboratory Manual. pp. E.37. 1989).
* Weigh 3 g of G-25 Sephadex into a 50 ml tube and fill it up with TE buffer pH 8. Add
NaN3 (0.02%) to it. Leave it overnight on the bench.

                                          18
* Use 10 ml syringes and put glass wool on the bottom of it, fill the syringe with the
equilibrated Sephadex


* Insert the syringe into a 15 ml disposable plastic tube. Centrifuge at 1600 g for
4 minutes at room temperature. Continue to add more resin and recentrifuge until the
volume of the packed column is approximately 0.9 ml and remains unchanged after
centrifugation


* Add 0.1 ml of TE buffer pH 8 to the column and recentrifuge as above. Repeat this step
twice more.


* The columns can be stored in the fridge wrapped with parafilm for several months.
(Make sure they do not get dry)


Adjust the volume of the labeled probe to 100 l with TE buffer and add to the column,
then spin it as above. The flow through will contain the labeled probe.


An aliquot of 1l of the labeled probe before and after the column purification is taken and
the CPMs are read. Readings of 0.1-0.4 X 106 cpm /l with a total volume of 90l are
commonly obtained.


Theory of gel chromatography:
This is a special type of partition chromatography in which separation is based on
molecular size.

A column is prepared of tiny particles of an inert substance that contains small pores. If a
solution containing molecules of various dimensions is passed through the column (Fig 1),
molecules larger than the pores move only in the space between the particles and hence
are not retarded by the column material. However, molecules smaller than the pores
diffuse in and out of particles with a probability that increases with decreasing molecular
size, in this way, they are slowed down in their movement down the column.


In Table 1 are listed the materials commonly used in gel chromatography and the
fractionation range.




                                          19
                 Fig .7: Separation of two molecules by passage through
                 a column containing particles of a porous gel. The molecules
                larger than the pores move more quickly than the smaller ones
               because the smaller ones move in and out of the pores.




              Table 1:




Ref: Freifelder, D. Physical Biochemistry. Applications to Biochemistry and Molecular
Biology. 2nd Edition. pp. 241. 1982.

                                            20
K     HYBRIDISATION OF THE LABELED PROBE TO THE MEMBRANE OF
      INTEREST CONTAINING THE RNA



Once the filter is baked or cross-linked, it can be stored at room temperature wrapped in
aluminium foil for weeks. If the filter is stored, it is important to rebake it before
hybridization (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A.,
Seidman, J. G., Struhl, K. Current Protocols in Molecular Biology, pp. 4.9.8. 1988).


Prehybridize the blot in the hybridization buffer for 1 hour with 50 g/l of denatured
herring sperm DNA, at the same time boil the probe for 10 minutes, put it on ice and quick
spin. Add the probe to the hybridization buffer (10 6 cpm/ml of hybridization buffer) and
place it in a rotating incubator overnight. All this process is done at 68oC for RNA probes
(45oC when using DNA probes and formamide containing buffers).
Hybridization buffer (Witchel, H. J., Maitland, N. J., Meech, R. W. Biotechniques 21: pp.
1024-1026. 1996; Church, G. M., Gilbert, W. Proc. Natl. Acad. Sci. USA, Vol.81. 1991-
1995. 1984).


7% SDS                     7g (slowly dissolve this amount in 50 ml of distilled water)
1% BSA                     1g (slowly dissolve the BSA and wait until it is completely
                            dissolved)
                                                              Final Concentration
1 M NaHPO4 pH 7.2          25 ml                              0.25 M
0.5 M EDTA pH 8)           200 ml                             1 mM
to 100 ml with dd H2O



Washes after hybridization to the probe (from Promega method in Riboprobe leaflet)
                                Final concentration
20 X SSC     6 ml               2 X SSC                22oC 5minutes
10% SDS      5 ml               0.5% SDS
up to 100 ml with dd H2O


20 X SSC     11 ml                 2 X SSC                    22oC 20-60 minutes
10% SDS      1 ml                  0.1% SDS
up to 100 ml with dd H2O


20 X SSC     500 ml                0.1 X SSC                  37oC 20-60 minutes
                                          21
10% SDS      5 ml                0.5% SDS
up to 100 ml with dd H2O


20 X SSC     500 ml              0.1 X SSC                  65oC 20 minutes
0.5% SDS 5 ml                    0.5% SDS
up to 100 ml with dd H2O


20 X SSC     2.5 ml              0.1 X SSC                  22oC 20 minutes
up to 500 ml with dd H2O


After the washes the membrane is wrapped in plastic paper and exposed to a
phosphorimager-screen from 1hour to overnight. Then the screen is scanned in the Storm
machine (or equivalent phosphorimager system). Alternatively the membrane can be
exposed to an autoradiograph.



Reagents:


20 X SSC


Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of water. Adjust the pH to
7 with a few drops of a 10 N solution of NaOH. Adjust the volume to 1 liter with water.
Sterilize by autoclaving.



M      STRIPPING OF THE MEMBRANE (QIAGEN PROTOCOL)



* Pour boiling 0.1% SDS solution onto the membrane and agitate for a few minutes


* Discard the solution and immediately add fresh, boiling 0.1% SDS solution


* Allow to cool to room temperature


* Autoradiograph the membrane to be sure that all the probe has been removed



(Notes:


                                         22
* If blot is boiled, the solution should contain at least 2 mM EDTA to prevent cleavage of
the RNA.
* The membrane should never be allowed to dry up otherwise the reprobing will not be
possible
* After the stripping procedure the membrane is wrapped in plastic paper and kept in the
fridge)




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