Running a 1% agarose gel
1. Gently remove the comb(s) from the pre-poured gel.
The gels have ethidium bromide in them- BE CAREFUL
and wear gloves!!
2. You should load 10 ul of cDNA/PCR sample + 5 ul of
dye. Also, in a separate late, load 10 ul of marker (NEB 1
kb marker)—this tells us what the size of our cDNA is (see
figure to the right). The smaller the fragment of DNA, the
faster it runs and the smaller DNA fragments will be
toward the bottom (see the marker scale again).
Cut a piece of Parafilm and tape it to your desk or
the flat side of a microfuge tube rack- see below. Pipette
the loading dye on the Parafilm, one dot per sample.
etc.
sample 2
sample 1
marker
The loading dye is made up at a 6x concentration, but if you are working with small
volumes, it is OK to exceed a 1x final concentration.
For example: 5 ul dye + 5 ul sample or
5 ul dye + 2 ul sample or
2 ul dye + 10 ul sample
Pipette your samples into the dye spots. Set your
pipette to the total volume. Pipette up and down to mix
the dye + sample.
_
3. Transfer each sample to the slot left after removing
the comb. This is “loading the gel” with your samples
(be sure your gels are in sample buffer with glycerol
and dye)—see figure to the right. Record the order of
your samples in your notebook (left to right).
4. Run the gel at 150 Volts (current should flow negative to positive – black on top, red
on bottom). This is
constant voltage (you can
also run gels with
constant current).
Proteins and DNA/RNA
are negative, so put the
red positive electrode
(anode) at the bottom of
the gel box. PHOTO: Gel
box on left, power supply
on right, comb in front.
+
1
Photography of the gel.
5.
5. Carry your gel (in the gel tray) to the UV light box. USE YOUR gloved fingers to
hold the gel in place or it will slide off!! Be sure not to drip buffer on the floor and don’t
let the gel fall to the floor (they are fragile).
EtBr IS A CARCINOGEN – WEAR GLOVES – CLEAN UP ANY SPILLS
IMMEDIATELY – FROM NOW ON TREAT THE GEL AS A BIOHAZARD AND
WEAR GLOVES
6.
Put on your UV glasses or face shield. Be aware of others around you, this is a
powerful UV light.
7.
Turn on the UV light and view your gel.
8.
If it looks good, take a picture with the digital camera. Look for the slot opposite
the handle and find the camera settings. Start
off with settings of an F stop of 16 (this is
how wide open the “lens” of the camera ismore open, better pictures with lower light)
and a 1 sec exposure. You may have to open
the lens more with a setting of F 8.Photo
below shows time of exposure (seconds) on
the top and the F stop lens opening on the
bottom.
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Appendix:
Pouring Gels:
1.
Measure 60 mL of 1X buffer (TBE) in a 500 mL Erlenmeyer flask.
2.
Add 0.6 g of agarose.
3.
Put a weigh boat on top of flask.
4.
Heat in microwave until boiling.
BE CAREFUL – ITS HOT – STEAM IS HOT
KNOCK OFF WEIGH BOAT LID BEFORE REMOVING FLASK FROM
MICROWAVE
5.
Swirl and re-heat until the solution is clear and you cannot see anything floating.
6.
Let solution cool so that glass is warm to the touch but not burning hot.
Alternatively, the flask can be placed in a 55 C water bath to cool and keep the
agarose melted.
7.
Add 6 uL of 10 mg/mL Ethidium Bromide to flask and swirl.
EtBr IS A CARCINOGEN – WEAR GLOVES – CLEAN UP ANY SPILLS
IMMEDIATELY – FROM NOW ON TREAT THE GEL AS A
BIOHAZARD AND WEAR GLOVES
8.
Set up your gel apparatus. Be sure ends are blocked off. Use the appropriate size
combs – be sure you have enough wells for all your samples plus markers and
controls.
9.
Pour the melted, but cooled agarose. Let the gel solidify. It will become opaque
looking.
10.
Turn the gel so that the wells are near the black electrode (the negative end).
11.
Pour enough 1X buffer (TBE) to just cover the top of the gel and fill the side
reservoirs. Too much buffer over the top will result in a longer running time.
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