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PCR RFLP is PCR product purification necessary?

pcr rflp

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3 replies to this topic

#1 harzan



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Posted 10 July 2013 - 04:14 AM

i want to find out SNP using PCR-RFLP, i want to know, should we need to purify the pcr product before rflp digestion?
i searched several protocol, where there is no any step for purification, but i have doubt about it.

#2 Trof


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Posted 10 July 2013 - 08:02 AM

You should you at least simple clean-up like precipitation, before you use your PCR product in restriction, because PCR buffer may not be compatible with the restriction enzyme requirements.
I mostly do a gel extraction, especially when I need to have nice picture, because remaining template gDNA gets restricted too and sometimes the smear from it spoils the image a bit.

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#3 pcrman



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Posted 10 July 2013 - 10:50 PM

Usually you don't need to do any purification. I once PCR-ed hundreds of DNA samples and subjected the products to restriction digestion without any purification. To minimize interference with enzyme activity by the salts in the PCR products, you should use as little (1-2 ul of 10 ul PCR reaction) as possible PCR product to give distinguishable bands on gel after digestion. To increase product concentration, you can use higher PCR cycling numbers.

#4 phage434



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Posted 11 July 2013 - 10:34 AM

I agree with PCRMAN. Most enzymes will work directly in the PCR buffer, but you can dilute in RE buffer if need be. You certainly don't need a fancy purification. Unless your template concentration is way too high, the template will be invisible on your gel.

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