PCR with radioactive nucleotides

Annette C. Hollmann ah690549 at mbcr.bcm.tmc.edu
Wed Nov 16 13:53:53 EST 1994


In article <01HJIHQKNE6Q002FLF at SCI.WFEB.EDU> ROSS at SCI.WFEB.EDU writes:
>
>We have been doing PCR with S35 nucleotides and have just found that
>the PCR block is heavily contaminated.  The manufacturer of the PCR
>says that S35 can go through the walls of the tube.  This is a little
>hard for me to believe.  I wonder if some tubes do not seal properly.
>In any case,  have other people had this problem and is there any
>way to avoid this type of contamination??
>
>					Alonzo Ross
>					Worcester Foundation
>					ROSS at SCI.WFEB.EDU
35-S probably cannot diffuse through microfuge tubes in the short time
required for PCR. That diffusion trick is more limited to nasty stuff like
125-I.
What is most likely happening is that volatile 35-S compounds are escaping
each time the tubes are heated. This has nothing to do with defective
microfuge tubes. You cannot put a completely airtight vial in a microfuge tube
or it would explode on heating or implode on cooling (remember that experiment
in elementary school where you put hot water in a plastic milk jug?)

If parafilm can withstand 95 C, it might be possible to reduce the
contamination by wrapping the tops of the tubes. This might trap some of
the volatile compounds.

If all else fails, use 1:1:1 hot water, ethanol, and PCC54 Plus to wipe
off the contamination (be careful not to use too much so as to avoid
shorting out the machine.

But beware: If the PCR machine is contaminated, some of the aerosol most
likely will also get into the air during a "hot" run, so don't hang around
near the machine.

Annette
ah690549 at mbcr.bcm.tmc.edu






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