We just bought the enzymes to investigate the polymorphisms in H pylori biopsies. When the student was preparing the G buffer to add in the restriction enzyme tube she added TAE instead ultra pure water in the Master mix. I know the TAE its acid and probably denatured the enzymes but I'm wonder if we removes the denaturation factor the enzyme can return to work properly.
Is anybody know how to removes this TAE from the restriction enzyme tubes?
Centrifugation? Clean up?
Any thoughts
Although my first recommendation is buying a new restriction enzyme given the low price of some of them, you can try dialysis using a membrane with pore size that impedes loss of the enzyme. The dialysis buffer should be the same for enzyme storage. You should change the dialysis buffer every 6h and perform at least 4 cycles.
TAE as in TAE-Buffer? Check your formulation of your buffer for more information on salt concentration and measure its pH to be able to infer anything meaningful regarding its possible denaturing property. I don't think there is any straightforward way to get rid of the TAE-Buffer once it's in there. I suppose you either purchase these enzymes again or check whether the TAE interferes with your downstream applications.
1X TAE is mild and may not affect enzyme activity. If your enzyme is over 10 kDa (supposed to be), you can use Abcam's spin column part #ab93349 to remove all buffer (dead stop at around 15 ul) in 20 minutes. Dilute concentrated enzyme with water and spin once or twice again to remove all residual TAE.
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