Exactly how to make a gel:
The large gel trays take 180-200ml of gel.
Think about what percentage gel you need to make.
Most agarose gels are~1%. 1% gel will show good separation of large DNA
fragments (5–10 kb). A 2% gel will show good resolution for small fragments (0.2–1
kb). In some cases a 3% or even a 4% may be required, though this is usually only for
products where you are trying to detect a very small size difference (as in microsat
amplification).
Let’s work with a 1% gel.
A 1% gel means that for every 100ml of 1xTAE buffer that we use, we will add 1g of
agarose. Since we’ll be making 200ml of gel, we will add 2g of agarose.
It’s best to weigh out the agarose first, and then add this to an empty microwavable
container before adding the buffer. If you add the agarose powder once the buffer is
already in the pot it’s more likely that you’ll get lumps of uncooked agarose in the
bottom of the pot.
Stir the uncooked gel mixture and then microwave on full power.
Normally 200ml of 1% gel will cook in around 3.5 mins. You can stir halfway though
cooking time. It’s important to keep an eye on the microwave to make sure that the
gel mixture doesn’t boil over. You can tell when cooking is finished as there will be
no undissolved agarose left in the container (uncooked agarose looks like little
bubbles or swirls in the liquid). When making a higher percentage gel, a rolling boil
rather than a frothing boil is a sign that all of the agarose has been properly cooked.
Take the cooked gel out of the microwave. Be careful and remember to use hot gloves
because there is a likelihood that as soon as you move the container it will boil over
onto your hands. Better safe than sorry.
Sit the gel mixture on the bench for a minute while you set up the rest of your gel
making kit.
Take a gel tray and put it in a gel caster. The gel casters lock a gel tray into place
when the little lever at the top points straight upwards. Lock the gel tray in place.
Use the circular bubble level to check that the gel tray is lying flat. If not, then adjust
the levelling screws that sit in the frame of the gel caster.
Place gel combs into the gel tray and make sure the screws sit in the little divots at
either side (see photos in “where to put your gel combs” attachment).
Your gel tray is now ready.
Now you can add the stain to your cooked gel mixture.
We use 5ul of EtBr for every 100 ml of gel, so our 200ml gel needs 10ul of EtBr.
Take care with the EtBr bottle!
If you just sit the bottom of the EtBr containing pipette tip in the hot gel the EtBr will
seep out because of the steamy action. If you try to squirt it out you’ll end up with
some gel mixture getting into the pipette tip and an annoying little bit of EtBr getting
stuck in there too.
Swirl or stir your stain/gel mixture well. If you don’t then you’ll end up with uneven
staining and so uneven fluorescing of product.
Gently pour the gel into the gel tray. If bubble form on the comb or in the main body
of the gel then you can push them to the side or pop them with a pipette tip.
Your gel is finished. It will take about 10 mins to set fully. A set gel will have an
opalescent sheen on the top and won’t squish when poked gently.
Remember to wash up your gel making container and tidy up after you.
Once set, remove combs gently by pulling straight upwards to reduce risk of tearing
gel around wells.
Tips for a prettier gel:
Thinner gels will give crisper bands than thick gels.
On the ethidium side:
Large gels take between 150-200ml.
Small gels take ~70ml.
The less buffer that covers your gel in the tank, the crisper your bands will be.
As long as your wells have buffer in them and the buffer is touching the sides of the
gel, your gel will run (regardless of how poorly covered by buffer it may look).
Cleaner buffer will give clearer gels than dirty buffer. Try not to change the buffer all
the time but if there are bits floating in the buffer or it smells a bit ‘milky’ then it’s
time to change.
High percentage gels (>2%) will froth less when cooking and look clearer when
imaged if the agarose is left to soak in the buffer (~15 mins or more) before
microwaving.