pcr dna purification for sequencing

Jan Hoolwerf hoolwerf at geocities.com
Mon Aug 7 07:53:47 EST 2000


Hallo felipe!

Op 04 Augustus 2000 schreef felipe wettstein (2:283/211.99) aan All:

 >> 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.

 fw> that is an interesting point, that with the not elongated
 fw> primers, i had no such problems, and i could read some probes
 fw> between 900 and 1000bp, and i was very lucky to do it like this,

"Luck" has something to do with it (and i'm not trying to make a nasty 
remark!)

We started sequencing without purifying the PCR products, works well 
many times. But not always and in these cases purifying did help a lot 
for the sequencing reaction. Often it was the difference between 
unusable and usable (not good <g>).

So, after a time we decided to purify all PCR products. It costs money 
(we use a kit from Pharmacia but others also do the job) buthaving to 
repeat the process was too much a hassle and sometimes even 
unacceptable when we really needed the result immediatedly.

 fw> , when you think the not elongatet primers are the only problem, i
 fw> guess you could try to optimise the pcr, like making a lot of 
 fw> cycles
 fw> and adding a minimum of primers.

Agreed, you should always do that. But the PCR we use is a compromise 
so it's probably often not optimal and then is purifying the PCR 
product very helpfull.


M.v.g., Jan Hoolwerf






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