Help with DNA acrylamide gel - leaking and errors during gel running - (Sep/12/2011 )
I've been trying to do a classic DNase footprinting assay using a 4% acrylamide gel. Two major problems keep occurring:
a) The acrylamide gel is leaking during setup. I use a 100ml solution, 10 ml of 40% acylamide/bis, 20 ml 5X TBE, 48 g of urea and water to 100ml . I add 700ul of fresh 10% APS and 30 ul of TEMED. Every time I set up my 40cm long plates with clips and gasket, the gel still leaks out of the bottom. How do I prevent this??
b) After the gel has set and is in my vertical gel unit, I get the E1 signal on my power supply when I try to run the gel. I've read that I should run 1-8V/cm of the gel so tried between 120-180 constant Voltage. It runs for a few minutes and then I get the "E1" error signal on the power supply every time. It seems to run longer before the error signal appears when I try higher voltages. I've tried using both 0.5X TBE and 1X TBE running buffer. Is it the buffer resistance? I've tried three different power supplies and get the same error no matter what. Any thoughts?
Also- during my most recent test, the bands for my DNA samples are very curved. I did a phenol chloroform extraction and ethanol precipitation, washed with 70% ethanol before resuspending in water. The samples seemed dirty, and did not resuspend very well. Is the curving due to high salt concentration in the sample? I also read that running the gel at lower voltage at the beginning can help prevent band curving, but since I get error signals constantly at lower voltage on the power supply I could not try this...
I think the 1-8 V/cm is actually for the distance between the electrodes, which, depending on the set-up, may be influencing how well the gel runs.
You can try pouring an agarose plug for the bottom of the gel - basically make a 1-2% agarose gel solution and pour it into a tray so as to have a couple of mm deep gel, Then clamp your plates together and stand them in the agarose. Allow to set, and your gels shouldn't leak.
Thanks for the hint about the agarose plug. I will try that this time.
I did try doing 1-8V/cm of distance between electrodes which for my vertical set up is about 35 cm. I'm not sure what's going on with the power supply and electrophoresis conditions. Could it be there is too much resistance in the gel so at a constant voltage setting the current drops too low to compensate for high resistance and falls below the minimum? If so, how do I fix this? I can't get the gel to run for more than a few minutes before the power supply Error 1 comes on and it stops running.
You could try running it at a constant amperage rather than constant voltage. For a big gel you may need to use a higher capacity power supply. Have a look around for what you can find on running sequencing gels.