Basic Subcloning Question
Austin So (Hae Jin)
haejin at netinfo.ubc.caX
Thu Jun 3 13:04:28 EST 1999
I never CIP'd either...mainly because if you CIP too long, there is a good chance
that you start chewing the ends off.
If you have a symmetrical fragment, then a 2:1 *molar* ratio of insert to vector
should favour the bimolecular reaction (insertion) versus the unimolecular
reaction (recircularization). For A/T overhangs, you should bring it up to 4:1.
IMHO, it is pretty trivial to do 20 minipreps (boiling) to check clones for
inserts and for orientation...that's a pretty good sample size (usually takes me
an 8hr day from start to finish). If you don't get it here, then odds are that you
will have to fart around regardless trying to get this insert cloned.
George Rutherford wrote:
> Quite right. Years ago, when I was a tech, I worked with a postdoc who was
> convinced (rightly or wrongly) that phosphatasing the vector was causing
> problems with ligation, so we just screened by colony hyb. Worked every
> time, and, frequently, more than 50% of the colonies were positive.
Austin P. So (Hae Jin)
University of British Columbia
E-mail: haejin at netinfo.ubc.ca
http://www.interchange.ubc.ca/haejin/index.html (under construction)
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