Agarose Gel Electrophoresis

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					                              Agarose Gel Electrophoresis
                                                                          Sandra, May 28, 2008


Comments and Overview
DNA fragments are separated in TBE buffer through agarose containing SyberSafe gel stain.
They are viewed on the DarkReader and can be photographed with the Digital camera.
For info about SybrSafe check out:
http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.46177.File.dat/SYBERSafe_FAQs
.pdf
Although SyberSafe is non-mutagenic in Ames tests, please use laboratory precautions. Use
gloves when handling the gel and remove gloves when touching common surfaces in the lab
such as light switches.
For info about the Dark Reader, check out:
http://www.clarechemical.com/
AND http://www.clarechemical.com/safe.htm
Gel apparatus is from BioRad. The manual is at:
http://www.biorad.com/cmc_upload/Literature/38717/M1704400B.PDF

Casting the Gel

   1. There are two tray sizes, 77 cm and 710 cm. For a single row of wells, use the smaller
      one. Position the tray in the gel caster between the rubber strips.
   2. Position the comb on the walls of the gel tray. For the older apparatus, adjust the comb
      with the aid of the thumbscrews so that it sits 1 mm above the base of the tray. The
      newer combs cannot be adjusted.
   3. Weigh out the appropriate amount of agarose directly into a 125 ml flask and add 1tris
      borate/EDTA (TBE).

   In choosing the % agarose (% is the grams agarose per 100 ml) use the following as a guide:
       For separating PCR products that are between 250 and 500 bp, use 2% agarose 1000
   (Invitrogen cat# 10975-035).
       For separating PCR products that are between 50 and 250 bp, use 3% agarose 1000.
       For distinguishing products larger than 500 bp use the cheaper agarose "Ultra Pure"
   (Invitrogen #15510-019) at 1% to 1.5%.
   For more info, see Fermentas' website:
   http://www.fermentas.com/techinfo/appendix/appendixtables1.htm

              For a 1% gel in the 710 cm tray, use 0.4 g agarose/40 ml TBE.
              For a 1% gel in the 77 cm tray, use 0.28 g agarose/28 ml TBE
              For a 1% gel in the wide min-sub cell, use 0.6 g agarose/60 ml TBE

   4. Microwave the agarose mixture on 50% power for 2 min. After 1:30, stop and swirl the
      flask Watch the flask in the microwave. If you see it bubbling, taking it out and swirl.
      Do not let the agarose boil.


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   5. Add 1/10 000 volume of SYBR Safe DNA gel stain (Stock in DMSO, Invitrogen#
      S33102; e.g. 4 l for 40 ml) and mix gently.
   6. Put in an alcohol thermometer. Swirl the flask gently while flowing cool tap water over
      the outside of the flask. Cool to 60C before pouring.
              Pouring the gel too hot can warp with gel tray.
   7. Pour the gel and ensure the comb sits properly. Pop any bubbles with a tip and let the gel
      polymerize for 20-30 min.
              A gel polymerizing time too long or too short can result in tearing of the wells.

Electrophoresis

    8. Carefully take out the comb. To prevent tearing the wells for gels 2% and higher, pour a
        little buffer on top and then gently pull out the comb. Place the gel tray into the gel box.
        Remember, the black connection goes behind the wells; "black in the back" .
    9. Submerge the gel under 2 to 4 mm TBE running buffer.
    10. Load the ladder(s). The 100 bp ladder is good for locating small fragments (< 500 bp).
        The 1 kb ladder is for locating fragments > 500 bp. Load 100 ng per mm lane width
        (works out to 4-10 l, depending on the type of comb used).
    11. Prepare your samples in 1DNA loading dye and load your samples. A quick way to do
        this is to spot the 6DNA loading dye on a piece of parafilm. Add the sample, mix and
        load.
    12. Connect the gel box lid. Run the gel at 80V until the purple dye migrates about 2/3 down
        the gel. View the gel on the Dark Reader.
        If desired, take a picture of the gel for you lab book. (See Digital Camera Use)
    13. If necessary, purify desired DNA fragments from the gel by excising with a razor blade
        and extracting the DNA. Remember to cut out bands on top of a protective glass
        plate. Preset the water bath at 55C.
    14. Dispose of the gel in biohazard waste. Wipe down the dark reader with water and a soft
        cloth to avoid scratching.
Unlike tris acetate (as in TAE buffer), tris borate buffer is much more stable during
electrophoresis. The same running buffer can be used up to 12 times before discarding. Rinse
gel equipment with water and then with tap deionized water. Let dry upside down on
absorbent paper.Do not let gel boxes sit with running buffer in them for extended periods.

Reagents and Solutions
In the past, we have tended to use Invitrogen products, as they are mostly available from
Biochemistry Stores. MBSU now sells Fermentas products, which tend to be a bit cheaper.
Remember to ask for a photocopy of the invoice to give to Chemistry Purchasing.

         Agarose:
           o "Ultra Pure" (Invitrogen #15510-019) OR TopVision LE GQ agarose
              (Fermentas# R0491)
           DNA size markers (ready-to-use):
           o GeneRuler 1kb DNA ladder (Fermentas #SM0313)
           o GeneRuler 100 bp DNA Ladder (Fermentas# SM0323)


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           o After thawing ladders, mix and aliquot. Store at -20C. Working aliquot can be
               stored at 4C.
       6 DNA loading dye: For your samples, use the sample buffer that comes with the
      ladder (6 DNA loading dye: 10 mM Tris-Cl pH 7.6, 0.03% bromophenol blue, 0.03%
      xylene cyanol, 60% glycerol, 60 mM EDTA).
       SYBR Safe DNA gel stain (Stock in DMSO, Invitrogen# S33102).


5  TBE (for 2 litres):
This stock can be made at 10 strength, but it tends to precipitate upon long-term storage.
                                             Final 1  working concentration
108 g Tris base
55 g boric acid                              89 mM Tris borate
40 ml 0.5 M EDTA pH 8                        2 mM EDTA



Taking Pictures and Printing with the PowerShot A640 Digital Camera:

     1.   Turn the camera on and ensure switch is in shooting mode.
     2.   Use P shooting mode.
     3.   Screw the camera on to the hood and position the hood on the dark reader.
     4.   Turn on the light.
     5.   If required, zoom in on the gel.
     6.   Hold the button down part way to allow the auto-focus action. Repeat until focus is
          satisfactory.
     7.   Without shaking the hood or camera, press the shutter all the way.
     8.   To review, switch to review mode and use the arrow buttons. Zoom also works for
          picture in memory.
     9.   To print a picture for your records:
            a. Connect the camera to the printer (Canon Selphy)
            b. Ensure camera is in review mode and turn it on.
            c. Wait a few seconds until display is in printing adjustment mode.
            d. "Function set" is also the ENTER button.
            e. Go to TRIM and centre the picture.
            f. Date/time stamp is always on.
            g. Go to print and press "Function Set".
            h. Important: Each picture must be tracked on the tally sheet (put a tick mark). A
                maximum of 36 pictures is allowed per ink cartridge.




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