Direct sequencing of PCR products

Dan Lasser 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,
>                    Simon.
> 
> 
Dear Simon:

	Try the Sequenase PCR purification kit, which is realatively new 
from Amrersham. 
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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