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bizarre digestion:)


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#1 myself

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Posted 03 January 2011 - 05:38 AM

I've used AcuI restrictase to detect a mutation. In a 500 bp PCR product it was supposed to detect a site ( in mutant only) and cut 150/350bp. it cuts my mutant suspect but also a negative control!!!! negtative control (in fact all 18 of them:) is for sure not matching the restriction site - i've sequenced it F and R. Time of digestion is moderate: 15 min to 1 h. I've added S-adenosinomethionine as NEB wanted it and used a correct reaction buffer. What on earth is going on!! have anybody encountered similar problem??
gls

#2 kajmak

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Posted 04 January 2011 - 01:30 AM

According to neb website, AcuI has a funny recognition site, with lots of N bases included. Is your mutation part of N sequence in AcuI site? Maybe the enzyme is not distinguishing between original and mutated sequences.
Unless you really need to digest that fragment with some enzyme donít bother with digestion especially is more than one person is using that enzyme.
As you said, sequence is good and just go ahead.

#3 myself

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Posted 04 January 2011 - 05:12 AM

The enzyme was brand new, and my mt is part of specific CTGAAG sequence ( but cuts further ). i need this digestion because its only small population of my cells that have the mutation, so i'd like to confirm what i've found on seqencing ( AS-PCR is not the way for me - have no good positive control)




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