发信人: biz (古怪), 信区: Biology
标 题: Re: how to avoid contamination when doing PCR?
发信站: The unknown SPACE (Sat Sep 16 03:20:37 2000), 站内信件

there are so many things that can go wrong in a pcr expt. your purpose would
be defined to carry out an expt without appreciable contamination rather
to figure out what has gone wrong.

typically if you want to find what's wrong, you would change one variables
(any buff, pcr station, even a pipette can be considered a single varibable)
at one time, repeat pcr expt and figure out what has gone wrong.

I would not do that though. you only have to figure out whether your own pcr
station has contamination or not and change all other things.

thus, talk to other guys in your group or other group, using their reagents
and pcr station, do another pcr expt using your own primer and theirs as
positive control. if it turns out that both work fine, the reagents you used
in your previous expt and/or your own pcr station are questionable. if
it turns out that your primer still gives products with contamination while
theirs doesn't, your primer oligo has got to have problem. check with where
you ordered oligo. it may result from a synthesis failure. use chromatograph
method, hplc for example, to purify your oligo, and use mass spectrometry
to verify its m.w.. if it turns out the batch of primer you used had problem,
try another batch until you make sure you are using the right primer you
want to use.

once you are sure that your primer oligo is right and it doesn't produce
undesired contamination products on a well-working pcr station, you can
use others' primer and reagents to carry out another pcr on your own pcr
station. this will tell you wheter you own pcr station is working fine.

if it turns out that your own pcr station is contamination free, then
change all the reagents you had used and prepare all anew by your
self. sometiems i don't trust technician or under helpers who routinly make
solutions for us.

hopefully, you can finally do pcr on your own station.

if you are not routinely use pcr, forget all i have said. simply do it
on others station and using their reagents. remebmer, your purpose is to
amplify your sequence, not to figure out what has gone worng with your
pcr station.


【 在 Cambridge (找寻心中的那缕阳光) 的大作中提到: 】
: my water control is always contaminated with DNA.
: when I get my DNA amplified,there is also a slight
: band in water control,which sucks.
: does anyone know a good way to avoid it?
: thanks.


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