sequencing chromosomal DNA

Tom Knight tk at
Wed Dec 7 13:13:56 EST 2005

Beth Rogers <erogers at> writes:
> We are trying to sequence chromosomal DNA using ABI's BigDye
> sequencing protocols and are getting no signals (plasmid and PCR
> sequencing works fine).  We have upped the DNA and primer amounts
> still with no luck. We are thinking of trying longer denaturation
> times(i.e. 10 mins), shearing, NaOh denaturation, DNAse nicking...
> Anyone have any experience with this that could offer some
> suggestions?  Thanks, ejr

You should increase the amount of DNA dramatically compared to what
you normally use.  You should cycle many more times (100x rather than
25x e.g.).  Make sure your primer is very specific and possibly raise
the annealing temperature to increase primer binding specificity.  See
the paper by Cheryl Heiner (1998) "Sequencing multimegabase template
DNA with BigDye terminator chemistry" Genome Res 8:557-61 PMID
9582199.  We did not use thermofidelase, but had an 800Kbp genome.
Signals were noisy, but good enough to determine primers, which we
used for PCR followed by resequencing.

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