(by hzhen from freeuk.com)
Thu Jun 28 07:40:19 EST 2007
Deitiker, Philip R wrote:
> Numbers in CFU
> - - - -CIP - No CIP
> No RE. 150 50 (Lost part of the gel during cutting)
> BamH1 0 57
> B&EcoR1 1 2
> B&E&H 0 0
> CIP decreases the level of reaction at least 40 fold based on inverse probability. There is no statistical difference between CIPing and No CIPing of double digest. Therefore it appears that CIPing is not neccesary and will be removed.
You are finding out something I discovered a long time ago. The problem
is that a lot of protocols in books just put in the CIP step without
even considering what the point of doing that is. For double-digested
DNA it is completely unnecessary. I personally think that the CIP step
is a big reason why a lot of people have problems with ligation, so they
ended up with using expensive ligation kits that don't give them any
flexibility in cloning.
Some people just follow protocols in books blindly. I've told a few
colleagues who had problem with ligation that they should just skip the
CIP step, they reacted with horror that I'd even think of not following
protocols to the letter.
> The conditions of ligation are appropriate, both buffer and enzyme are still working, also the DH5 alpha cells are still viable and transformation capable (even diluted 4 fold, with rather small amounts of DNA).
The viability of the cells should be taken as granted (if they are not
viable, then there is something seriously wrong with the way you
prepare, use or store your cells). You should get a lawn of cells when
you transform 1 ng of uncut plasmid.
> The comment made about cutting bands from gels and using an agarase free purification kit. This appears to work since the unRE digested DNA was capable of producing ~100 CFUs per ul of ligation reaction the ligation also was +/- efficient with 30% reclosure of BamH1 cut vector. So gel purification
> did not appear to interfere with either.
> What interferes with ligation without insert 1. CIPing 2. Double digests with gel purification.
> 3. Triple digests with gel purification.
> Not neccesary to test with kinase at this point, since kinase activity on double digest will not restore ligation, and because CIPing is not neccesary on double digest.
> The assay shows that with controls and careful band cutting it should be possible to determine if ligation occurred based on a control reaction with only on enzyme (No insert), and a control reaction with no insert as well as reaction with insert.
> Thanks for the help, at least I know what the confidence ranges are on these.
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