Madhadron:Cruft: Difference between revisions
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* Use 30s instead of 1 minute for the three stages of PCR in general. Strongly binding primers will bind just fine in 30s, extension for 30s in 1 minute will reduce nonspecific large stuff, and 30s at denaturation will destroy less of the polymerase at each cycle. | * Use 30s instead of 1 minute for the three stages of PCR in general. Strongly binding primers will bind just fine in 30s, extension for 30s in 1 minute will reduce nonspecific large stuff, and 30s at denaturation will destroy less of the polymerase at each cycle. | ||
* Can try adding 5M DMSO if there's still a lot of nonspecific junk. | * Can try adding 5M DMSO if there's still a lot of nonspecific junk. | ||
* Can PCR straight out of ''E. coli'' and ''M. smegmatis'': take a scoop of colonies from a plate, suspend in 100μL PBS+Tween, boil for 5-10 minutes, and use 1-2μL of that as the template in PCR reaction. | |||
Revision as of 13:58, 22 February 2007
My own personal brand of labelling obsessiveness:
- DNA shall hav a light green dot on the top of it's tube if it is a maxiprep; bright orange if it is primer; and I haven't had to assign a miniprep color yet.
- Chemicals will be labelled on orange tape if sterile, and on green if they are not.
- E. coli stock will have a dark green dot on the top of its tube. M. smegmatis will have dark blue.
Neeraj's advice for revising my PCR:
- Titrating Mg, DNA, etc. is for when you don't get any bands; no good if you have too many bands.
- Use 30s instead of 1 minute for the three stages of PCR in general. Strongly binding primers will bind just fine in 30s, extension for 30s in 1 minute will reduce nonspecific large stuff, and 30s at denaturation will destroy less of the polymerase at each cycle.
- Can try adding 5M DMSO if there's still a lot of nonspecific junk.
- Can PCR straight out of E. coli and M. smegmatis: take a scoop of colonies from a plate, suspend in 100μL PBS+Tween, boil for 5-10 minutes, and use 1-2μL of that as the template in PCR reaction.
