pcr dna purification for sequencing

felipe wettstein felipe.wettstein at ioez.tu-freiberg.de
Mon Aug 7 07:24:13 EST 2000


hallo jan

>  >> 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, 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. those of you
that work with abi probably do mostly dye terminated sequencing pcr's,
there i guess it would really be a bad idea to take an unpurified
aliquote of pcr as template for the sequencing pcr.
of course i agree also that purifiing is nesessary when there are
multiple bands and when the reaction did not work properly.

>  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!)
:-))

> 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.
i agree, but since for the few times i did like described above it
worked fine, i will continue till it does not work any more, and then i
will remember your word.

enjoy your day

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






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