Friday, July 3, 2009

The PCR I ran yesterday was a replicate of one SS did last week. We used the same ingredients and thermocycler. Which means nothing's wrong with the stuff, but I'm using them improperly.

PCR variables and controls:

1. Used approximately 165ngs of lab stock MAP7 DNA. I've used this to transform competent cells before, so it shouldn't be weird. I could try some dprA::kan chromosomal DNA I have, but SS got a nice band with MAP7 last time.

2. Used Hi crp primers, which are SS's. She used them with MAP7 and got a product, so there's nothing wrong with the primers. Well, actually, maybe there is, since MAP7 and RecJ primers only showed up faintly and MAP7 and ExoI primers didn't show up at all when SS ran the PCR.

3. The gel electrophoresis equipment is fine, because the 1kb ladder runs nicely.

4. Is it the thermocycler, somehow? But the first time I tried this, I used the Thompsons', and that didn't work. Plus, as I've already said, SS used the same cycle on the same machine and got results.

5. Is there a specific way that I should be adding the ingredients? I always Taq last, but I don't have a certain order for the other ingredients. Should I be vortexing or centrifuging them? We don't have a lot of Taq left; maybe it's really old?

I've noticed that my products always seem to be very watery when I mix them with the gel dye. I can't explain this very well, but when I mix chromosomal DNA with the dye, the whole blob of liquid turns blue (and it remains a blob), but when I mix the PCR product with dye, the dye stays at the bottom and the blob turns into a flat lake, almost. I don't quite remember, but I ran the gel for SS's PCR, and her products seemed to remain a blob. I know that doesn't really confirm anything (yes, we know PCR's not working for you, Rhena).


1 comment:

Garry Potter said...

Man, didn't know about this blog until now. I read your latest post and was lost but I guess I'll have to start from the beginning. Looking forward to it.