Basic Subcloning Question
Lemee at ibm.net
Fri Jun 4 10:03:45 EST 1999
I totally agree on the lab technique here regarding CIP (I don't know about 'farting
around' all day...). Ratios (and quality: always geneclean and/or etoh ppt before you
ligate) of insert/vector during ligation is a better way to go at it. I usually
guestimate the ligation at 1 vector:3insert overnight at room temp, zap and plate DH5a
from gibco in the evening next day (expensive but you get your clone every time, I
always wonder at people making their crappy home grown comp. cells and then using
qiagen columns to miniprep the plasmids out) . I then miniprep 8 to 18 colonies
(depending on how good looking the plates are). The boiling minpreps take less than
1h15 total time, 1 hour digest , 45 minutes gel run: in one morning you got your
answer, works every time !
"Austin So (Hae Jin)" wrote:
> 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)
> Biotechnology Laboratory
> 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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