Digestion of PCR-product

Lab 356 se15 at le.ac.uk
Wed Sep 29 08:01:15 EST 1999


Eduard Post wrote in message <37F1F72A.8C951F2B at biol.rug.nl>...
>Hello,
>
>I'm having problems in my restricition digest of fluorescent-labeld
>PCR-product!
>
>There is a large undigested portion, I can't get it to be digested.
>
>this is my digestion mix:
>5 microliter of PCR-Product (purified)
>10 units of AluI or HhaI
>1.5 microliter BSA
>
>the total volume is 7.5 microliters of mix.
>
>Please does anyone have the same problems or does know a solution?
>
>eduard
>


Glycerol in your enzyme may inhibit the reaction.
As a general rule you should always have the final volume of the
reaction at least 10 times the amount of enzyme added.
In your case (I assume your enzyme is at 10U/ul) you have only 7.5X volume

I would do the reaction in a 20ul final volume and also include reaction
buffer as
Krimer Alejandro suggests!

Steve







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