Optimal Gel Electropheresis Protocol 2-14-13

2015-01-13

azim58 - Optimal Gel Electropheresis Protocol 2-14-13

Optimal Gel Electropheresis Protocol 2-14-13
protocol can also be found here:
"S:\Research\Cancer_Eradication\Discovering tumor specific
antigens\Protocols\Gel Electropheresis\Optimal Gel Electropheresis
Protocol 2-14-13.docx"

Optimal Gel Electropheresis Protocol
-Determine the concentration of gel you would like to make.
--For expected fragments around 100-1000 bp a 1.5% gel is best.
--For expected fragments around 1-4 kb a 0.8% agarose gel is best.
--For expected fragments around 4-6 kb a 0.6% agarose gel is best.

-Determine the number of lanes, size of lanes, and volume of gel you
would like to make.
--The volume of solution for the medium gels should be 100 mL
--The volume of solution for the large gels should be 350 mL

-Prepare the gel by mixing the correct amount of agarose with the correct
amount of liquid. Cover the top of the flask with ceram wrap and poke
with holes to prevent evaporation of the liquid and therefore
concentration of the gel. Microwave the gel mixture for 1 min 30 s. Do
not add ethidium bromide to the gel solution for optimal results since
this could later result in a "shadow" on the gel. Let the solution cool
for 10 min (a curved gel that is higher at the edges will result if
really hot buffer comes into contact with the cool casting chamber).
--The 350 mL solutions are best prepared by splitting the solution in 1/2
and preparing each half in a separate beaker. So for a 0.8% gel mix 2
solutions of 1.4 g agarose with 175 mL buffer. For a 1.5% gel mix 2
solutions of 2.63 g in 175 mL buffer.

-Pour the gel solution into the casting chamber. Allow the gel to
solidify for 1 hr.
--If making a 350 mL gel, the two solutions can be poured together at the
same time into the casting chamber.

-Perform pre-electropheresis to equilibrate the gel with the buffer. This
can be done by mixing 1% EtBr with buffer (3 uL 1% EtBr into about 450 mL
buffer for a 100 mL gel). Then place the gel in the electropheresis
chamber, and add buffer so that the buffer is just about 1 cm higher than
the gel (too much buffer can later result in overheating above the gel
and therefore a "rainbow" on the gel). Connect the wires to the gel so
that the negative black wire is on the side where the DNA will later be
loaded so that the negatively charged DNA will be pushed away from the
negatively charged wire (sometimes the wires must be dipped into the
buffer in order to get them to apply a voltage). Then run the gel without
any sample in the gel at about 150 V (medium 100 mL gel) or 300 V (large
350 mL gel) for about 1hr45m or 2hr for large 350 mL gel to make sure the
ethidium bromide passes through the whole gel (a UV picture can be
obtained to confirm that the ethidium bromide front has passed through
the gel). Small bubbles should rise from the bottom to the top of the
chamber as the voltage is being applied. Then turn off the voltage. The
gel can be stored in the cold room in buffer if desired.
--Note that higher voltages for longer times can result in heat at the
surface of the gel which can lead to smearing of the bands.

-Before loading the DNA you can replace hot buffer with fresh buffer
without any ethidium bromide added, but this is not completely necessary.
If the gel was stored in the cold room with buffer, allow the gel to come
to room temperature before loading any DNA samples. Load the DNA samples
while keeping track of how much sample (volume and concentration) is
loaded into each lane. Usually 12 uL of ladder is loaded. Note that even
as little of 20 ng of DNA can theoretically be visualized on an
electropheresis gel.

-Increase the voltage to a level appropriate for the size of the gel.
Allow the gel to run for about 1 hr. Note that longer run times and/or
higher voltages can lead to heat at the surface of the gel which can
result in smearing and a "rainbow".
--For a medium 100 mL gel the optimal voltage is around 68 V.
--For a large 350 mL gel the optimal voltage is around 172 V
--Note that for DNA that is not purified such as DNA from a restriction
digest or a PCR reaction run the solution into the gel very slowly (e.g.
maybe about 20-30 V for 10-20 min until the sample is in the gel), and
then later increase the voltage in order to prevent a "rainbow" in the
gel.
--Note that higher voltages for longer times can result in heat at the
surface of the gel which can lead to smearing of the bands.

-Visualize the gel. We use the "ChemiDoc" to visualize the gel.
--Login to the Quantity One software. Select the scanner "Chemidoc XRS".
Make sure the black slider at the top of the Chemidoc is in the middle
position. Select "UV transillumination". Use EPI white and live exposure
to move the gel into place (paper towels can be placed below the gel (not
underneath) to make sure that it doesn't slide everywhere). Adjust the
zoom and focus if necessary (a transparent ruler can be used to get the
numbers of the ruler in focus if desired). Choose autoexposure or manual
exposure (usually around 0.5-2 s for the manual exposure is appropriate).
Exposure times that are too long can result in bands of bright and dark
light across the image. Note that UV light should be turned on for
several seconds (2-3 s) before starting the exposure for an image to
allow the light to fully come on and stay constant. The first time you
expose the gel the software may give you some message so you may want to
go through this process and get the message without actually exposing the
gel to UV light at first, and then go back and do the real exposure with
the UV light. If bands are hard to make out on the gel after obtaining a
good image, it may be necessary to adjust the brightness, contrast,
and/or invert the colors in the image.

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