S35 thermal cycler contamination
finney at Frodo.MGH.Harvard.EDU
Fri Jul 23 16:38:01 EST 1993
In my alternate identity as chief scientist of MJ Research, I've become
concerned about reports of S35 contamination of thermal cyclers. What
follows is a summary of what we think is going on, some questions that
we would like to ask anyone who has information, and some suggestions
for controlling and cleaning up contamination.
Radioactive contamination has been reported by people using S35 (but not
P32 or P33), principally for cycle sequencing. What appears to be
happening is that the thiophosphate nucleotides break down chemically,
yielding a sulfur-containing product that is volatile at high
temperatures. One possibility is H2S, but there are a number of others.
Whatever it is, the radioactive compound can get out and contaminate
things in two different ways. First, it can get out around the rim of a
tube. This is likely to be a problem only if you are using an oil-less
configuration (an oil overlay should prevent volatilization) and the
amount of contamination should vary with the brand or lot of tubes.
Second, it can get out through the wall of the vessel. The
polycarbonate plates sold by MJR and other vendors are not permeable to
liquids, but they are slightly permeable to gasses (such as water
vapor). Thus we would not be surprised if some S35 could go through the
walls of the plates. Thin-wall polypropylene tubes are NOT permeable to
water vapor, so we would be somewhat surprised if S35 compounds could
get through them (but we can't absolutely rule it out either). These
tubes are usually used without oil, so we would rather suspect leakage
around the rims.
We don't know anything about the chemical identity of the offending
molecule (ask NEN or Amersham!) but there may well be different amounts
in different lots or brands of label and the amount may increase with
the age of the label or the number of times it's been freeze-thawed. A
few years ago, there was a similar problem with S35-methionine for
metabolic labeling of proteins. Does anyone know how this was resolved?
I would like to ask anyone who has done S35 cycle sequencing to drop me
a quick note (by email; I will post results) answering if possible the
following (and anything else useful):
1 Did you experience contamination?
2 Where was the contamination (block, lid, gloves, etc.)?
3 How severe was the contamination?
4 What type and brand of reaction vessel did you use?
5 Did you use an oil overlay?
6 Did you use oil in the thermal cycler block?
7 What was the temp. and time of denaturation, and # of cycles?
8 What was the brand of the label?
9 How old was the label?
Finally, what to do about it. This is preliminary, and includes some
helpful suggestions that were previously posted.
1 Use P33. Yes, I know it's expensive, but it will definitely solve
the problem and it works great.
2 Put your cycler in a hood. If the S35 can get out of the tube,
you're breathing it, and that's not good.
3 Keep denaturing times and temperatures to a minimum. This will limit
both volatilization and the pressure inside vessels.
4 For plates, use a small amount of oil in the thermal cycler block.
This will limit the amount of gas (including water vapor) that gets out.
An easy way to do this is to dip the underside of a plate into a small
dish (pipet tip box top?) of mineral oil before putting the plate into
5 Use oil overlays. I know it's inconvenient, but it's better than
losing your radioactive license.
6 This is speculative. Add something to the reactions that will sop up
the guilty compound. Possibilities include dithiothreitol (DTT),
oxidized DTT, and Fe II or Fe III. Be judicious with the iron, as it
can attack DNA, especially in the presence of EDTA.
As for decontamination of thermal cyclers, we don't know of anything
that works well. Detergents apparently have little value, so the S35
may well be covalently bonded. Reasonable candidates are mild bases
(NH4OH), reducing agents (DTT), or oxidizing agents (bleach, hydrogen
peroxide). Don't use bleach on bare metal (all MJR cyclers have
anodized blocks, so bleach can be used _occasionally_). We would love
to hear from anyone who has something that works.
finney at ochre.mgh.harvard.edu
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