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Summary: S35 contam. and cycle sequencing

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Mon Aug 9 03:01:45 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 
>Mike Finney
>Mike Finney

For removal of S35 from aluminium blocks (Corbett Machine) I have used
Decon 90 at about 5% (Selby product) which although says can affect
nonferrous metals but did not seem to do any damage (apart from make the
wells shinier).  I just added the solution, heated to about 70*C for 10 min
(trying to avoid S35 vapour) and aspirated.  Repeating a few times and then
washed well with water several times.  This depleted the counts from
~500cps to less than 5 in about 5-6 washes.
As I have also said previously I am convinced that the contamination does
come through the walls of the tube.
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"I know that you think that you think that you understand what I am saying."
"But what I am saying is sometimes not actually what I mean."

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