LESSONS IN PARANOIA
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LESSONS IN PARANOIA
Procedures for making solutions:
- With few exceptions all solutions must be made RNase free.
- Sterile plasticware can be assumed to be RNase free.
- Autoclaving will not kill RNase. Baking at >250 C for 5
hours will.
- No utensils should ever go into chemicals - one can use a sterile
pipet from wrapped plastic.
- No chemicals should ever be returned to the bottles after it's
been removed.
- All bottles must be washed on long dishwasher cycle and then
baked in drying oven approximately 5 hours.
- Caps must be autoclaved - let sit in fresh DEP-H2O approx.
20 minutes first (see below).
- Weigh out chemicals onto baked glass beakers, etc...
- Avoid contact with hands - a potential RNase carrier - wear
gloves as necessary.
- Stirring bars must also be treated. Again place in fresh DEP-H2O
for approximately 20 minutes before autoclaving. Never pick up
stirring bars with hands - use gloves.
- For solutions without amines, etc.. use H2O directly from
DI tap into baked glass cylinders, etc...
- Once solution is made, pH'd, etc... filter sterilize through
nitrocellulose.
- Aliquot into sterile glass bottles <2/3 full.
- Add DEP (diethyl pyrocarbonate) to 0.1%. Shake and let sit
approx. 1 hour.
- Autoclave for approx. 23 minutes on liquid.
- DEP will react with Tris, EDTA, etc... anything with reactable
nitrogens.
- Therefore, treat the DI-H2O with DEP first, i.e., make H2O
0.1%; let sit for approx. 30 minutes. Then, continue as in #11
without re-adding the DEP.
- DEP is unstable to air and H2O - it should be kept on ice
and capped when it is being used.
- For stirring bars, caps, etc... make 0.1% DEP-H2O.
- Stock solutions labelled DEP-H2O are autoclaved H2O pre-treated
with DEP.
- DEP is destroyed (breaks down into CO2 and H2O) by autoclaving.
- DEP reacts with proteins and hence destroys RNase activity
and restriction, etc... of enzymes.
- Bulk solutions need not be treated in this fashion. These
include:
- all media
- all Southern selections
- DNA gel buffers
- After removing stock solutions from common storage area:
- Note on list if more is needed.
- Decide if this will be used as an RNA solution and label bottle
accordingly or sequester as you choose.
- Only freshly opened pipets or autoclaved pipet tips (clean
pak pipet tips can be autoclaved as they are - we have autoclave
tape) should go into these bottles and wear gloves as needed.