pcr dna purification for sequencing

kerstin.hoef-emden at uni-koeln.de kerstin.hoef-emden at uni-koeln.de
Mon Aug 7 14:07:53 EST 2000


Hi,

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

>>  >> 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.
> i discussed with some collegues and they told that there is a big
> difference between dye terminator sequencing pcr and labeld primers. we
> use labeld primers and so,

Indeed, reactions done with labeled primers are less sensitive to
concentration changes in chemicals or experiments with unpurified PCR
products. 

> the primers from the backwards reaction do
> not disturb the detection in the sequencing gel, because they are not
> labeld and therefor not visible for the sequencing machine. 

They might eat up the dNTPs and ddNTPs, but they will not cause ambiguous
sequencing reactions.


Regards,
  
Kerstin


-- 

	kerstin.hoef-emden at uni-koeln.de				 






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