LESSONS IN PARANOIA

LESSONS IN PARANOIA

Procedures for making solutions:
  1. With few exceptions all solutions must be made RNase free.
  2. Sterile plasticware can be assumed to be RNase free.
  3. Autoclaving will not kill RNase. Baking at >250 C for 5 hours will.
  4. No utensils should ever go into chemicals - one can use a sterile pipet from wrapped plastic.
  5. No chemicals should ever be returned to the bottles after it's been removed.
  6. All bottles must be washed on long dishwasher cycle and then baked in drying oven approximately 5 hours.
  7. Caps must be autoclaved - let sit in fresh DEP-H2O approx. 20 minutes first (see below).
  8. Weigh out chemicals onto baked glass beakers, etc...
  9. Avoid contact with hands - a potential RNase carrier - wear gloves as necessary.
  10. 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.
  11. 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.
  12. 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.
  13. DEP is unstable to air and H2O - it should be kept on ice and capped when it is being used.
  14. For stirring bars, caps, etc... make 0.1% DEP-H2O.
  15. Stock solutions labelled DEP-H2O are autoclaved H2O pre-treated with DEP.
  16. DEP is destroyed (breaks down into CO2 and H2O) by autoclaving.
  17. DEP reacts with proteins and hence destroys RNase activity and restriction, etc... of enzymes.
  18. Bulk solutions need not be treated in this fashion. These include:
    • all media
    • all Southern selections
    • DNA gel buffers
  19. 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.