Gel
Electrophoresis of your PCR reactions
Pouring
agarose
gels
1.
Seal
both
ends
of the gel tray with tape or stoppers/dam.
2.
Place
comb
in
slot at the end of the gel tray.
3.
Pour
melted
agarose
into the gel tray until the
gel is about 5 mm deep. Let the
agarose harden, which should take 5-10 minutes. Don’t
touch/move
your
gel
until it’s hard. In
the meantime, prepare your PCR
reactions for electrophoresis.
Electrophoresis
PCR
reactions
1.
Using a
clean tip for
each reaction, pipet 2 µl gel orange loading dye (OJ) into each
PCR reaction
tube.
You will
load
both your PCR reactions and standard DNA ladder into the gel. A
DNA ladder has many pieces of DNA of
known size so you can compare the DNA from your PCR reaction to the
ladder. In
this way you can determine what size your PCR product is. Two or three
groups
might share a gel, but only one molecular weight marker is needed per
gel.
2.
In your
lab notebook, record where you loaded each
sample, (PCR reaction(s), DNA ladder). Record the lane number and the
name of
the sample. Be certain to have the information of where the other
groups added
their samples.
3.
When your
gel has
hardened, carefully push tray out of holder or remove the tape. Gently
wiggle
the comb out of the gel.
4.
Place the
gel tray with
the gel in an electrophoresis box. Make sure that the wells are aligned
with
the negative end of the box.
5.
Pour TBE
buffer so it
fills the electrophoresis box and just covers the gel.
6.
Beginning
with the left
side of the gel load your samples in the wells. Load the DNA ladder
into the
middle well.
Extract
the
sample from the tube and making sure that the sample is visible in the
very end
of the tip. Place the pipet tip directly over, or just inside of, the
first
well. Slowly push down on the plunger to release the sample. Once the
sample is
expressed, remove the tip from the well and buffer before releasing the
plunger.
Do not
be
overly concerned with getting more than the very end of the tip over,
or into,
the well opening as the loading dye has glycerol added which will cause
the
sample to sink into the wells.
7.
Continue
to add your
samples until finished. Be sure to change tips each time and record in
your lab
notebook, which sample was loaded into which well.
8.
Now run
that gel! Plug the electrodes into your
electrophoresis apparatus (red to red, black to black), being careful not to bump your gel too
much. Make sure one more time that your
wells
and DNA samples are at the negative (black) end of the electrophoresis
box.
9.
Plug the
power source
into an outlet and set the voltage at 75 to 90 V.
10. Let the gel run until the dye migrates
about 2 cm from far end of the gel (about 20-25 minutes).
11.
Turn off
the power supply, disconnect the electrodes,
and remove the top of the electrophoresis apparatus.
12.
Carefully
remove the gel and place it on a weigh tray
with your group name on it.
Staining
Gel
with
Ethidium
Bromide -
Instructors will do the staining for you!
1.
Using
gloves, remove the
plastic from the ethidium bromide (EtBr)
sheet and place the ethidium bromide paper on the gel. Add a few drops
of
buffer to ensure transfer of the stain and gently rub the paper with
your
fingers to make sure it is contacting the gel all over.
2.
Stain for
about 10
minutes. Discard EtBr papers in
the red EtBr trash. Throw gloves
in this same trash bag if you actually handled EtBr contaminated
materials.
3.
Put the
gel in the UV
transluminator. Close the door and turn on the white light to make sure
that
the gel is properly aligned. Turn off the white light, turn on the UV
and push
“Live” to see the gel. Adjust light using + or -.
4.
Take a
Polaroid picture
of your gel; tape Polaroid picture into lab notebook and record all
relevant
information with the image.
Materials
Needed
Ethidium
Bromide
sheets – Ethidium bromide is carcinogenic. Do not allow
students to
handle EtBr sheets or gels stained with EtBr
Ethidium
bromide waste bag
Staining
trays
Equipment
necessary
20 ul micropipette
Gel
boxes and casting trays
Electrophoresis
power supplies
Gel
doc analyzer/UV transilluminator