help with sequencing of pcr-products

Chris LaRosa clarosa at biocomp.unl.edu
Fri Mar 31 12:11:00 EST 2000



skladny at geocities.com wrote:
> 
> hi anyone out there
> 
> actually I have started with IRD-sequencing of PCR products using a
> LI-COR
> unit. The primer is labeled with IRD 800 and sequencing of plasmid-DNA
> works very well. PCR products often can't be read the first 50 to 80 nt
> after the pbs, and some PCR products give very strong stop-signals in
> all four lanes (no difference if cleaned up by gel electrophoresis or
> unpurified PCR-product used). Though the PCR conditions seem to be very
> stringent it might be an idea to use even more stringent PCR conditions
> to get rid off the unwanted shorter products?   
Hi ,   we routinely cycle sequence off pcr products using the ABI
system.
Our lab considered buying the Licor system but we ruled that out because
it really is not possible to routinely sequence off pcr products with
the Licor chemistries.  Further More it is too expensive if you need to
make primers for alot of different products.  The only way I am aware of
doing it , without a lot of optimization, is to TA clone the 
pcr products into a plasmid.   

Have you tried contacting Licor directly? They claim it is possible to
do it , but I have not heard about anyone who has had sucess. However,
we have been approached by sales reps who claim it can be done.    (By
the way we work in Lincoln Nebraska, where the Licor manufacturing
facility is... I interviewed for a job their several months ago, and at
that time noone could tell me that sequencing off pcr products was
routine).




More information about the Methods mailing list