Making high concentration agarose gels - A request for advice (Apr/27/2007 )
I have been trying to cast 4% agarose gels, but whenever I do so using the usual techniques that work when making lower concentration gels -- i.e. microwaving agarose powder in buffer solution until it melts -- I encounter problems. Specifically, when the solution boils, a lot of bubbles are formed, but it is very difficult to get rid of them because of the viscousity of the solution. I have tried letting the solution sit on a heated plate to wait for the bubbles to disperse, but even with heating the gel starts to solidify, I suppose because of the high concentration of agarose; so I was forced to pour my gel with some bubbles still in it.
Can anyone offer me advice on how to cast high-concentration agarose gels properly -- that is, without bubbles? I would be very grateful!
Heat the buffer first, then add the agarose a little at a time with mixing. Let the mixture hydrate for 30 minutes or so. Finally, heat to dissolve. You would probably do better with a 2.5 to 3% metaphor agarose gel.
Can anyone offer me advice on how to cast high-concentration agarose gels properly -- that is, without bubbles? I would be very grateful!
ur problem is almost common for all, casting the high persentage gels. whenever ur casting agarose gels of more than 2% you first weigh agarose powder first, pour it in the conocal flask, then add respective amount of buffer. then heat it in microwave oven. while heating u stirr it but donot let it for over heat because water will evaporates from the buffer and the gel will become thick and u will get more bubbles. after melting the agarose switchoff the oven and keep the flask inside oven for 1 to 2 minutes then take out and wash under running tap wate for 3 to 3 mins jut to cool the flask. then pour it in the mold and remove the bubbles with the 200μl tip immediately after pouring. u will get the clear gel.
you heat it too fast and high temperature?
i normally cook my gel in medium-low temperature... cook for few minutes, take out swirl the flask to let the heat spread evenly, put back into microwave oven again and medium low for another few minutes.
But sure you need to add more buffer to avoid excess evaporation.
i'm doing this for all my gels (high and low percentage of agarose). heat it slow and put more buffer.
hope my idea can help you
Good luck!!!
i normally cook my gel in medium-low temperature... cook for few minutes, take out swirl the flask to let the heat spread evenly, put back into microwave oven again and medium low for another few minutes.
agree with you, medium-low temperature is favored in these cases
I would also recommend heating at low temperatures (I normally use about 350 Watt for my 3,5% gels). I tried adding the powder after heating but that did not work well: Very often I got chunks of Agarose which didn't disolve at all.
It is very important that the flask is cover with saran wrap or other plastic cover that can be use in microwave (NO parafilm!!!!!). then heat the mix in increments of 10-15 seconds and swirl (no shake), let it cool down for a few min and cast it (is better to cast it a little bit hot, if no will solidify before finish) and with a pasteur pipet move the bubbles to the sides.
I recommand NuSieve: agrose 3:1 or low melting temperature agarose for your case