Rules of thumb for sequencing cloned PCR products?

J. Justin McCormick mccormi1 at cranium.com.msu.edu
Thu Sep 5 17:31:56 EST 1996


In article <50ebhg$93s at ukwsv3.ggr.co.uk> Fraser Dr N J <njf16154 at ggr.co.uk> writes:
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>From: Fraser Dr N J <njf16154 at ggr.co.uk>
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: Re: Rules of thumb for sequencing cloned PCR products?
>Date: 2 Sep 1996 10:05:36 GMT
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>bjm10 at newsstand.cit.cornell.edu (Bryan John Maloney) wrote:

>>It may be a rule of thumb, but I know that an amplification that a post-doc
>>in our lab did was worse than that.  She used a mixture of Taq and Pfu and
>>still got two deletions.  We know that they're deletions because she was 
>>amplifying a synthetic sequence originally assembled from oligos.  
>>Furthermore, a different clone from the same amplification ended up not
>>having the deletions.  The deletions were confirmed by one machine and
>>two "by hand" sequencings.

>Having made numerous synthetic genes in the past, the deletions etc observed
>are not necessarily due to the amplification step. If you directly clone
>synthetic sequences assembled from oligo.s without any amplification step, you
>often find 'mutations'.

>Neil.





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