Agarose 'rafts' blocking the top of sample well - Difficulty pouring a 3% gel (Jun/18/2007 )
Hi,
I think this may be a silly mistake I'm making here, since it's been a while (~6mos) since I've poured gels. Recently, whenever I pour an 3% TBE-agarose gel and allow it ~20min to cool & solidify, I remove the comb to find rafts of agarose left behind in many of the wells. The chunks look like they're floating freely above the bottom of the well, and are difficult to force get out of the well without damaging the walls of the sample well. It's causing problems because I cannot load my samples as consistently as I'd like, since the effective volume of the well into which the sample settles is reduced, I'm basically overloading the well with a very small sample (6ul) volume. I'm using same agarose lot & %, same TBE formulation, same comb, etc - that Ive used in the past, so I'm rather puzzled what is different now. I'm thinking I've forgotten something really simple. I'm wondering if anyone has encountered this, and if so, can offer a remedy. Cheers!-JAH
I think this may be a silly mistake I'm making here, since it's been a while (~6mos) since I've poured gels. Recently, whenever I pour an 3% TBE-agarose gel and allow it ~20min to cool & solidify, I remove the comb to find rafts of agarose left behind in many of the wells. The chunks look like they're floating freely above the bottom of the well, and are difficult to force get out of the well without damaging the walls of the sample well. It's causing problems because I cannot load my samples as consistently as I'd like, since the effective volume of the well into which the sample settles is reduced, I'm basically overloading the well with a very small sample (6ul) volume. I'm using same agarose lot & %, same TBE formulation, same comb, etc - that Ive used in the past, so I'm rather puzzled what is different now. I'm thinking I've forgotten something really simple. I'm wondering if anyone has encountered this, and if so, can offer a remedy. Cheers!-JAH
Hi Jah, I also get bits of agarose that are present in the wells when I go to load my samples. However, they are fairly small and when I run my index finger along the top edge over the wells of the gel, they lift out easily. In your case I would make sure the combs are clean and dry, pour the gel soon after (2-4 minutes) you dissolve the agarose, and try pre-heating the comb.
It may sound obvious, but you can also try removing your comb very slowly, and watching it as it comes out... just before it's about to come out and 'break the suction' caused by lifting the comb, go really slowly. Lift it out by pulling it out straight up, not to the side, or front or back. Hold both sides of the comb. Ok, enough of the obvious! You can also buy some silanizing material to make your combs come out easier. You can buy some that is designed for combs, or you can buy some of that 'rainoff' or 'rain-x' for car windshields. Lastly, is the agarose touching the top of the comb in between the wells? If so, maybe you can pour your gels a little shallower.
Cheers,
Scott.
The problem is very simple you are no waiting enough. 20 min is to few time for a 3% gel, let stand between 30-40 min to make sure it polymerise well. Always keep combs very very clean and when taking it out use steady upward force and in 1 movement. Ahhhh and before forget, always take the comb out inside the tank with buffer (this will help to mantein the wells open).
you should be more patient to make 3% agarose gel
dissolve the agarose powder in buffer properly ,
i use oven to heat the agarose and buffer to dissolve and i mix them by hand after every 30 sec. to avoid bubble mix very very slowly. if i use 200 ml buffer then it takes about 3-4 mins to dissolve the agarose properly
after dissolve, mix properly and let it settle down and pour it very carefully in gel plate. if any bubble arise remove it with a help of pippet. and 20 min is not enough ..........you should wait 40 min atleast