Problem when making PAGE gel - (Aug/01/2005 )
When I pull out the comb from the gel, there are always some threads of gel inside the wells. The threads sometimes block my loading.
Could anyone give me some advices? Thank you very much.
be shure the gel is fully polymerised when pull the comb out. we also first assamle the gel apparatus, fill it with running buffer and then pull the comb (under buffer). after this, we use a syringe with a relative small needle to press cautiosly some running buffer into the well and therby cleaning the well of residual gel rest...
I used to have this problem all the time. I take a gel-loading tip and blow air into the wells to clear them out, making sure all air gets removed before running it. If it's copious, I'll try to suction it out with those same tips using a 200 ul pipetteman (apply tip to junk, suction, slowly remove - it'll peel off, leaving the wells intact).
I found out later that the problem is mainly caused by polymerizing the gel too quickly. I reduced my APS and TEMED concentrations by 25%, and I rarely see junk in the wells anymore. It could also be caused by a dirty comb.
what we do is flush the wells with water using a squeeze bottle. Then just shake the gel once or twice to get the water out. I usually repeat this 3-4 times and the well clears up. I also agree with the lower TEMED and AMP idea.
Thank you for your replies!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
You can also try to put silicon cream (like surfers use for their boards) on the comb. I have heard, it should come out just easy and without threads left. But I have never tried myself. I usually just use a syringe and the running puffer to blow everything out of the pockert. Works good.
I typically pull my comb out under running buffer- and then I take a gel loading tip and insert it into the gel and push any threads to the side. Also helps straighten any wells if they sagged.
I do the same as Pria and also I swab the comb(gel plates and spacers too) with ethyl alcohol.