pcr dna purification for sequencing
hoolwerf at geocities.com
Mon Aug 7 07:53:47 EST 2000
Op 04 Augustus 2000 schreef felipe wettstein (2:283/211.99) aan All:
>> It is possible to do it without purification, but usually
>> is worse and reactions do not go so far, when compared to purified
>> products. This becomes most obvious when the PCR yield is
>> because a lot of non-elongated PCR primers are left, competing with
>> the sequencing primers for the resources in the sequencing
>> If you just want to identify the product, do it without
>> if you want to read 900 to 1000 bp, it is better to purify the PCR
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
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> 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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