IUBio

sequencing phage dna

john augliera auggie at MIT.EDU
Fri May 31 23:20:31 EST 1996


Would anyone know of a protocol for sequencing phage dna.  I am running a
Perkin-Elmer/ABI 377 automated sequencer and have been using their Taq-Fs
Ready Rxn Kit for dye terminator sequencing.  I have an investigator who
would like to sequence lambda clones directly and avoid the subcloning step.
She has told me that they have manually sequenced such clones and would like
to take advantage of the automated sequencers throughput capacity.  We have
tried some test samples from her lab with varied success.  Only one sample
produced sequence data but signal was very low and overall quality was not
great.  I have been using the standard thermal cycling protocol recommended
for the Taq-FS kit. I was wondering if an alternate protocol should be used
for phage dna and would appreciate any info. anyone has on a method they have
used which routinely works.


P.S. I am using the PE 9600 thermal cycler



Let me thank everyone in advance for any advice offered.


                                                                Sincerly,
                                                                John Augliera
                                                                auggie at mit.edu





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