direct sequencing of PCR products

ilyas m khan khan at hollywood.u-net.com
Fri Jul 18 18:26:37 EST 1997


In article <5qiqn1$kgo$1 at bignews.shef.ac.uk>, J.Atkinson at shef.ac.uk (J
Atkinson) wrote:

> Any hints for successful sequencing of PCR products?
> I want to sequence a 100bp PCR product without cloning.  I have been 
> told a method exists which uses DMSO in all solutions, anyone know 
> anymore.  How small can the primer be?  Any tips to get sequence as 
> close to the primer as possible.
> 
> Thanks
> Jen


if you're using automted sequencing, you can directly sequence the product
if your automated sequencer fluorescent PCR kit is of the old type, i.e
not this new fangled error correction stuff. if you dont purify your
intial template with a clonetech C-50 column (or similar) you will miss
the first 20-30 bp, as these are either unreadable or more usually
wrong!if you're not using automated sequencing then Maxam Gilbert is
usually recommended, but its a bastard to do, and you have to get hold of
hydrazine, which is now banned in the UK as the IRA used to use it to blow
up things unconnected with molecular biology.


                                          hope this helps

                                             ilyas



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