S35 PCR

Claus Jorgensen claus at tamarin.demon.co.uk
Wed Jul 14 12:58:20 EST 1993


In article <9307130039.AA17665 at cscgpo.anu.edu.au> Klaus.Matthaei at anu.edu.au writes:

>G'Day Netters
>
>Some months ago there was a question about S35 PCR and the possibility that
>the label may be going through the tubes and contaminating the block.  We
>have recently obtained a 96 well cycle sequencer (apologies to Cetus) that
>uses thin-wall tubes or thin-wall 96 well plates.  The system does not use
>oil but sealed tubes.  Although P32 PCR goes without a hitch (i.e. clean
>block), when S35 is used the block inside the wells (but nowhere else)
>becomes contaminated (200-500cps) if either of these tubes is used.  Our
>only conclusion could be that the S35 was going through the tube walls.  
>Amersham now inform us that S35 is volatile at high temperature (and of
>course it should be , its sulphur).  We are still trying to establish
>whether the S35 is from breakdown of S35dATP or from any free label that
>may be in the preps that are sold.  We are still in conference with
>Amersham.  Presumably P32 is also volatile but we don't have the same
>problem so my money is on free S35 in the preps.  
>My point to the net is, has anyone else seen this problem?  Does this also
>happen with standard thick-walled PCR tubes.  And CAUTION please check your
>system. 
>
>I will keep in touch as things progress.

Thanks for bringing this subject up.

Your theory about volatile S-35 getting through the tube walls can explain
the problems we have encountered in our lab with S-35 PCR.  
We are using a 96 well machine with microtiter plates and another machine 
with thick-walled tubes. Both blocks on the two machines are S-35 
contaminated. Even after using non-corrosive Decon on the blocks, we can't 
get rid of the contamination. I would therefore be delighted if someone could 
put their decontamination procedure forward.


Cheers,

Claus Jorgensen
Dept. of Immunology
AFRC Babraham Institute
Cambridge CB2 4AT
United Kingdom



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