pcr dna purification for sequencing

kerstin.hoef-emden at uni-koeln.de kerstin.hoef-emden at uni-koeln.de
Fri Aug 4 08:55:52 EST 2000


Hi,

felipe wettstein <felipe.wettstein at ioez.tu-freiberg.de> wrote:

> i would not purify the pcr product, since in your pcr reaction mix there
> are only things that you need also for sequencing-pcr, like taq,
> template and nucleotides. in our MWG manual for the licor sequencer
> there is a sequencing reaction described as follows:

It is possible to do it without purification, but usually background is
worse and reactions do not go so far, when compared to purified products.
This becomes most obvious when the PCR yield is suboptimal, because a lot of
non-elongated PCR primers are left, competing with the sequencing primers
for the resources in the sequencing reaction. If you just want to identify
the product, do it without purification, if you want to read 900 to 1000 bp,
it is better to purify the PCR products.


Regards,

Kerstin


-- 

	kerstin.hoef-emden at uni-koeln.de				 






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