Deitiker, Philip R
(by pdeitik from bcm.tmc.edu)
Mon Jun 25 15:16:58 EST 2007
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: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of ChenHA
Sent: Thursday, June 21, 2007 2:19 PM
To: methods from magpie.bio.indiana.edu
Subject: Re: Ligation
Deitiker, Philip R wrote:
> I am trying to clone several fragments into a derivative of E4276 a
> derivative that has a BamH1 - - - - EcoR1 cloning site.
> I have had successful ligations in the past, I managed to make several
> clones of the nascent vector and they cut true to the restriction
> But I have had no success cloning 6 Bgl-II -Insert -- EcoR1 constructs
> into this vector. Even following protocols suggesting 10 fm of vector
> and 30 fm of Insert, all I get back are reclosed vectors.
> *Bgl-II has compatible sticky ends.
Do you suspect that the protein may be toxic to the cells?
> Originally the situation was very bad, many vector colonies, but I
> worked on the digestion to reduce the number of colonies to about
> 1/10th, approximately 10 vector closures per 10fm of DNA.
> The ends of the Insert contain
> C-A-C-G-G-A-G-A-T-C-T-T-G-G - - - - -
> According to NEB this should be enough overhang.
> as this
> . . . T-C-C-A G-A-T-C-T-T-C-C - - - - - cut to 100%
> The EcoR1 site is the same thing
> C-G-G-G-A-A-T-T-C-A - - - - - - - - - - (My insert)
> t-c-g-a-G-G-A-A-T-T-C-C - - - - - - - - (NEB, lower case = single
> stranded overhang.)
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 other issue is transformation. Many here say use 1 ul of DNA for transformation.
> I don't know if this works or not, but I have linear increase in
> clones from 1,2,4,8 microliters. More than 8 uls of ligation mixture
> did not result in more clones and eventually clones declined.
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.
> So my thinking here is that if 1 ul is adequate my ligation is really tanking.
> The only transformations are the vector, somehow unclosed after hours
> of digestion, near complete digestion in 1 hour with either enzyme
> (when tested independently and CIP for several hours.
> The vector is grown, Minipreped, restriction digested (monitoring
> timepoints by gel) CIP, promega SV PCR cleanuped, prior to reaction.
> As for the Insert.
> After restriction digest I clean the DNA up with gene clean turbo,
> more recently because the mid range ligation is 30 fm I did a EtOH
> precipitation completely dried the DNA and resuspended with 1/10X warm
> ligation buffer. So there should this is now three purifications removed from the last Gel. Still no success.
> Is it worth making new primers and starting from scratch? is it a
> common primer that a certain sequence of overhand works and a very similar overhand sequence does not?
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.
> Thanks in advance.
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