Help me answer this curious student - Many questions on techniques (Dec/06/2009 )

Hello All,

So this new project student is very curious and asking me tons of questions. I just don't have concrete answers for his questions. Some of the questions are -

1. Why should you freeze PCR buffers after use? They are just salt solutions afterall so why should they not be stored at room temperature?
2. What will happen if I keep extension step of PCR for 2 mins for 1kb fragment (when Taq manufacturer recommends 1min/kb)?
3. Why should you freeze oligos / primers after your use? Why can't they stored in fridge at 4°C? Do they not degrade at evert freeze-thaw cycle?
4. Is it possible to prepare your home-made PCR master-mixes and store them in freezer? if yes, how long do they last? (I know you get ready made master mixes but I have never tried home made master mixes)
5. If you contaminate your hands with Ethidium bromide, how long does it take to wash it off? (I usually wash my hands with soap and as far as I believe, it cleans EtBr but I am not very certain.)

Many more but these are the only ones on top of my head.

Thanks for your comments.

0. Hire this kid. Sounds as if you might have already figured that out.
1. If you could keep it sterile, this might be ok. Bacterial growth happens everywhere, and can change buffer composition and add unwanted DNA. Buffers typically also contain things like Tween or Triton X-100 or BSA all of which can provide nutrients to sufficiently enterprising bacteria.
2. Usually all that happens is that you come back later for your results; but sometimes it can affect things, if you have multiple binding sites for a primer, some of which are spaced further apart.
3. You can store primers at +4, but two things: (1) bacteria (2) DNAse contamination from e.g. fingers. I tend to store primers in TE rather than water or Tris, which is the usual preferred method, on the theory that if you look at the amount of EDTA put into the PCR mix from adding primers (which I add from 30 uM stock) then the change in Mg++ concentration is minimal.
4. Yes, certainly, there is no magic. The major difficulty is stability of proteins in dilute solutions, often addressed with a carrier such as BSA. But PCR enzymes are unbelievably robust.
5. 1-2 days in my experience. I think mostly the skin wears off, sort of like henna. Gloves. Not so important in this case, but make sure she knows about things like acridine orange.