pcr dna purification for sequencing

felipe wettstein felipe.wettstein at ioez.tu-freiberg.de
Fri Aug 4 11:26:47 EST 2000


> > 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.

that is an interesting point, that with the not elongated primers, i had
no such problems, and i could read some probes between 900 and 1000bp,
and i was very lucky to do it like this, because in our lab there was
only easypure to clean the pcr product out of the agarose gel, and i did
not like this kit (dont ask me why, i just did not like it).
and then, when you think the not elongatet primers are the only problem,
i guess you could try to optimise the pcr, like making a lot of cycles
and adding a minimum of primers.

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-------| * ~. -felipe-wettstein----------------------------------------






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