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Based on some of the replies here and the reagents I had available I did a set of test ligations.
The RE digest were BamH1, BamH1 & EcoR1, BamH1&EcoR1&HindIII, None The treatment was CIP 1 hour and no CIP during the last hour of digestion. Total of 8 reaction variations.
The material was gel purified on a 1%A/TBE gel with 0.075ul/ml of EtBr. The bands were cut under longWL UV handheld.
Initial finding, All bands cut to completion, except the no RE bands (CIP and no CIP) both lanes had about 50% of the product in supercoiled vector and relaxed vector.
In this case only the supercoiled vector was cut.
The bands were purified with Qbio 101 gene clean III kit using 12.5 ul of glass milk for each, there was some inperfection in the cutting which unfortunately corrupted one of the controls (No CIP No enzyme).
The resulting DNA was 12.5 ul of which 5 ul of each was added to a new tube a mixture of 5 ul containing T4 ligase and 2X ligation buffer. Since I am running out of DH5alpha I used 12.5 microliters of DH5 alpha and 1.5 ul of ligation mixture.
The cells were incubated for 30 minutes in a 4'C block
2 minutes at exactly 42'C and 2 minutes on 4'C block again.
250 ul of SOC was added incubated on a rotator for 45min followed by plating O/N at 37'C
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.
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 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.
From: [Only registered users see links. ] [mailto:[Only registered users see links. ].indiana.edu] On Behalf Of ChenHA
Sent: Thursday, June 21, 2007 2:19 PM
To: [Only registered users see links. ]
Subject: Re: Ligation
Deitiker, Philip R wrote:
Do you suspect that the protein may be toxic to the cells?
Just an idea - since you are ligation BglII to BamHI, then what you can do is add BamHI to the ligation mixture. The religated plasmid will be digested, but not the one with insert. (This is a useful trick in blunt end ligation, but may be applicable in your case). Something to think about, but
for directional cloning you really don't need to do this.
You actually don't need to do CIP treatment to start with because you have two different ends, so using CIP is rather pointless, just causing more problem. In my experience CIP should be avoided unless absolutely necessary.
The standard advice is to use a volume equal to 5% of the competent cell, i.e. if you use 100 ul of cell, then use 5 ul of the ligation mixture. This is to avoid having too much contaminants which may affect the transformation efficiency. However, I regularly use 10 ul of ligation mixture in 100
ul of cells with no problem, and as you found, you can use more without problem.
Normally I just add 7-8 extra bases to save me the bother of checking.
Here's what I do with ligation -
Digest, and then I always gel purify my digested DNA, and always use low melting point agarose (this may be unnecessary, but since my ligations nearly always work, there is no reason to change the way I do it, even if just passing it through a column is faster and should give reasonably clean DNA).
Don't bother with EtOH precipitation.
For ligation, use 10-100 ng of vector DNA, and 1-3X insert in 20 ul of reaction. Heat DNA mixture at ~50 degC for 5 minutes, cool, then add ligase and buffer. Leave ligation O/N in fridge (why do you use warm ligation buffer?) then transform.
If you are preparing a vector DNA fresh, always do a test ligation with no insert - this is to check the background religation. Often I get 5-20 X more colonies for those with insert compared to those without, and you can then be sure that most of the transformants has the plasmid with insert.
Even if you have an equal number of transformants compared to the test ligation without insert, a good proportion will have the insert, just need to do an extra screening step (say, by PCR).
The most important thing is having good competent cells. Transformation efficiency of over 10^8 is ideal. And no CIP.
Lastly, someone posted something about cloning methods without ligation, you can try those if this continues to be a problem.
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Deitiker, Philip R wrote:
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 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.
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