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Question about Double Digestion followed by PCR amplification

Restriction digestion Enzyme PCR Sequential digestion

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2 replies to this topic

#1 hotstuffdb22



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Posted 07 September 2013 - 08:59 AM


I'd really appreciate some assistance with the following question:  is it possible to sequentially double digest genomic DNA followed by PCR amplification for the region of interest?


While it makes intuitive sense, my reason for doing this is that I am working with a transgene in a mouse study that is difficult to detect by normal PCR.  Thus, my strategy is to increase the PCR efficiency by first setting up a sequential digest with KpnI and ScaI.  I'll ethanol purify after each digestion and then will run the PCR.  Do you think this will work?




(BTW- I just started the first digest, so any feedback will help me out a lot).

#2 bob1


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Posted 07 September 2013 - 02:12 PM

It doesn't really make all that much sense, other than it would chop the DNA into shorter fragments and so potentially make it more accessible to the polymerase.  You may well find that it is more important to get the primers and reaction conditions exactly right.

#3 student47



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Posted 19 September 2013 - 07:53 AM

not an expert but i also think that you might cut other sites in the whole genome (if it is whole genome) which might have same restriction sites. may be if you can digest and then run on gel and extract product of your size, if you find it enough, and then use that as template for PCR. this still has a chance that other sites with similar restriction cut can produce a similar size product (if whole genome) but still if you can find good concentration enough after digest and gel recovery , along with you selected primer specificity, it should definitely work. good thinking though.. :) 

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