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:
>From: Fraser Dr N J <njf16154 at ggr.co.uk>
>Subject: Re: Rules of thumb for sequencing cloned PCR products?
>Date: 2 Sep 1996 10:05:36 GMT
>Organization: Glaxo Wellcome
>Message-ID: <50ebhg$93s at ukwsv3.ggr.co.uk>
>References: <32231624.7189 at ic.ac.uk> <505dp0$suv at newsstand.cit.cornell.edu>
>Content-Type: text/plain; charset=us-ascii
>X-Mailer: Mozilla 1.1IS (X11; I; IRIX 5.3 IP22)
>X-URL: news:505dp0$suv at newsstand.cit.cornell.edu
>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'.
More information about the Methods