S35 PCR

Victor Jongeneel vjongene at isrec-sun1.unil.ch
Wed Jul 21 06:40:15 EST 1993


Claus Jorgensen (claus at tamarin.demon.co.uk) wrote:
: In article <9307130039.AA17665 at cscgpo.anu.edu.au> Klaus.Matthaei at anu.edu.au writes:

: >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.

: 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.

I don't have specific suggestions as to a decontamination procedure, but
both of you may want to check 33P as an alternative to 35S for thermal
sequencing.  It behaves like 32P chemically (i.e. no oxydation problems,
high Km for DNA polymerase) but emits beta particles with an energy similar
to 35S, giving similar quality sequences.  I am willing to bet that 33P
labeled nucleotides will not produce breakdown products that can diffuse
through thin-walled tubes, and I can testify that sequences look just as
good as with 35S.

Hope this helps!
--

C. Victor JONGENEEL                      vjongene at isrec-sun1.unil.ch
Ludwig Institute for Cancer Research
Chemin des Boveresses 155                       FAX: +41-21-653-4474
CH-1066 EPALINGES                               Tel: +41-21-316-5994
Switzerland                 "Where the cows moo and the people vote"



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