Contamination woes...HELP!

Marieke R. Koedood Zhao rkoedood at bu.edu
Mon Jun 1 11:49:44 EST 1998


I got that in my early PCR days. First PCR beautiful, next one all were 
positive. What aved me after that was not using the same pipets for setting
up the reaction and analyzing the product. Also set up my reactions 
on a different bench (or even lab) as the one where I analyze the PCR product.

Marieke

ccc6 at Lehigh.EDU wrote:
: Hi!
: I've been having immense problems with contamination in my PCR reactions.
: Recent results suggest that I have plasmid contamination in the reaction mix.
: Over the past few months, I have tried everything I can think of to get rid of
: the contamination.  I usually get one or two good sets of reactions and then
: all of my negative controls start showing up positive again.  We use the
: following for PCR setup to avoid contamination:
:     MilliQ water in sterile tubes
:     Filter tips for addition of all reagents
:     Set up under hood (w/o blower on) that is UV irradiated when not in use
:     Wipe surfaces and pipettors with RNAse Away solution that claims to get
:         rid of DNA contamination from surfaces
:     Clean out holes of thermal cycler with RNAse Away solution and Qtip before
:         placing tubes into unit

: I can't think of much else to try to get rid of this problem.  I've started
: over with brand new reagents numerous times, but the contamination always
: comes back after a few sets of reactions.  I've also had reactions with all
: new reagents come up contaminated.  Occasionally I get clean results
: with already used reagents, too.  I can't predict when the contamination is
: going to show up.

: Any suggestions would be appreciated.
: Thanks in advance,
: Christine




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