I'm working on a gel extraction of a 40kb piece of DNA from Lonza's SeaKem gold agarose (from Pulse Field Gel Electrophoresis). It was a 1% gel, the band's already been cut out and 0.7mL of 1x beta-agarase reaction buffer (10mM Bis Tris-HCl, 1mM EDTA) was added to the tube with the band. Its been sitting in a water bath for hours holding a little above 90oC with very little of the gel slice actually melting. Lonza's manual lists the melting temp for a 1.5% gel as above 90o C.Does anyone have any advice on how to speed up the process?
You need to boil most agaroses to melt them completely and this would damage the DNA. Have you added beta-agarase enzyme to the gel slice and incubated at the appropriate temperature? If your enzyme is alive, you should have no trouble digesting the gel. I cannot remember if borate inhibits beta-agarase. Check your gel running buffer for inhibitors.
Hi,
Unless the DNA is very difficult to obtain, I would strongly recommend to repeat the whole thing with low-melting temperature agarose.
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