how to melt agarose, and keep it melted? - (Oct/12/2005 )
I need to melt agarose beads (20 ul), and keep them melted at room temperature. My beads contain DNA, which I use for PCR (one bead per reaction). After PCR, I need to load it for gel electrophoresis, but my reaction solidifies too quickly and I can't load it into the wells in time.
Does anyone know what the composition of Buffer QG (used in QIAGEN Gel Extraction Kit for melting solidified agarose) is?
Alternatively, does anyone have any suggestions of other solutions I can make up from regular lab chemicals that I can add to my agarose beads to keep them in liquid form at room temperature?
You want to run your DNA in liquid support? I mean, first, I'm not aware of any way to keep the agarose liquid at RT, and secondly, even if it remains liquid at RT, how do you intend to run the gel, since it wouldn't have gelled.
I think you could use an oven to keep the agarose warm so that it dosen't solidify so fast. Any reagent u want to add in it - take out the flask, add it, put it back in the oven. Maintain about 60 degrees, it wouldn't hurt your hand, nor will it let the agarose solidify. Once everything is done, simply take it out of the oven, pour it and let it solidify (at RT of course).
Hope this helps.
Most gel extraction kits rely on a high molarity chaotropic salt solution (e.g. sodium iodide or guanidine isothiocyanate) at a pH of 7.5 or less (e.g. 4 - 5 M salt in Tris pH 7.5). I'm pretty sure Qiagen's uses guanidine isothiocyanate.
A question -- since your DNA is in agarose, and you obviously have a gel extraction kit, why not just gel extract the DNA from the beads and use the extracted DNA as PCR template?
Thanks for the suggestions.
Homebrew: I guess I could. My DNA was intially embedded in the agarose bead for bisulfite treatment (methylation studies), so they end up modified and single-stranded. I've found that leaving them in the beads preserves the DNA for longer. So I just plop one bead into the PCR mix when I need it.
But you gave me an idea. I could gel extract my PCR products at the end of the reaction, and then run that on a gel to see what they look like. If I get the band I want, I can purify that by cutting out the band and gel extracting again.
Thanks again for the help
Hope your experiment is going well.