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Summary: S35 contam. and cycle sequencing

Michael Finney finney at Frodo.MGH.Harvard.EDU
Fri Aug 6 20:15:56 EST 1993


In independent posts and responses to my query about radioactive 
contamination caused by cycle sequencing with S35, I got a total of 7 
useful experiences.  I will smmarize below:

For those just tuning in, a radioactive breakdown product of S35 
nucleotides seems to get out of tubes and microtiter plates.  This has 
caused headaches for many people.

The breakdown product is most likely H2S.  One can easily draw acid- or 
base-catalysed mechanisms for the following reaction:
 O             O
OP=S + H2O -> OP=O +H2S
 O             O
The best evidence came from Barton Slatko at NEB.  He sees a diffuse 
band at about 40 bp only in S35 reactions that have been thermally 
cycled.  The diffuseness is consistent with low molecular weight, and 
the position is consistent with the fact that H2S is a weak acid.

There was no obvious effect of brand or age of label.

The concensus for the route of contamination is through the tube walls.  
Only two people (Susan Forsburg and Barton Slatko) reported low or no 
contamination, and both were using thick-walled 0.5 ml tubes.  On the 
other hand, one person (Stephan Regenass) reported contamination when 
using only thick-walled 0.5 ml tubes (National Scientific) with an oil 
overlay, so use of these tubes is no guarantee.  Perhaps there is 
variation between brands or lots.  Bill Warren reported that Perkin 
Elmer tells him there is lot-to-lot variation among their 0.2 ml tubes.  
Also, all thin-walled tubes are fragile and "craze" when squeezed; this 
could certainly make matters worse.

For preventing contamination, the preferred method is to switch to P33; 
for those who canUt afford it, using thick-walled tubes may help.  No 
one reported trying weak oxidizers in the reaction mix to react with the 
free H2S.  If anyone wants to try I think the most practical one would 
be cystine (i.e., oxidized cysteine) which is cheap from Sigma and 
shouldn't interfere with anything.  Several people suggested using 
activated charcoal to absorb the contamination, as is done in tissue 
culture incubators.  This might help somewhat, but you can't put 
charcoal between the tube and the cycler block, which is where most of 
the contamination builds up.

As for cleaning up, the only new suggestion was from Annette Hollmann, 
who uses the detergent PCC-54 from Pierce.  I called Pierce technical 
service, and they said PCC-54 would corrode aluminum.  So for cyclers 
with bare aluminum blocks (Perkin Elmer, Ericomp), it is definitely out.  
For MJ Research cyclers (which have anodized blocks) it can be used 
occasionally, but all detergent should be thoroughly removed after 
cleaning.

Mike Finney
Mike Finney



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