Direct sequencing of PCR products
dml1 at columbia.edu
Mon Feb 27 15:55:36 EST 1995
On 21 Feb 1995, Simon Dawson wrote:
> Does anybody have good experience of direct sequencing of PCR
> products? If anybody out there does have a good method they would
> like to share, I would be more than grateful if they could email me
> a copy or post it to this newsgroup.
> Thanx in advance,
Try the Sequenase PCR purification kit, which is realatively new
I have used it and had success with multiple PCR fragments.
The advantages are that tedious subcloning is avoided. If you have a nice
clean product, you can also avoid gel purification.
The kit basically employs DNAse I and shrimp alkaline phosphatase to
remove the PCR oligos and dNTPs. Then the sequencing reactions follow the
same basic protocol as for Sequenase sequencing .
It works but there are certain caveats: your primers MUST have a "good"
design and anneal at 55 degrees. Specificity is critical as is
avoidance of "short" oligos.
I have been truely satisfied with the results. Good luck.
Dan Lasser, Columbia U. Sch of Med
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