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Yet another cloning problem

Yet another cloning problem - Molecular Cloning Forum

Yet another cloning problem - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 10-14-2011, 05:17 AM
Pipette Filler
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Default Yet another cloning problem



So I have a plasmid that contains an insert with NdeI and BamHI into pet3a. My aim is to do an N-terminal deletion. So I have designed a Forward primer with NdeI site and a reverse primer with BamHI site. The BamHI has AAAAAA as extra and NdeI has AAAA as extra. The two have good compatibility and the PCR reaction goes well, where I get the right sized PCR product as my insert.
To get my vector, I cut the original plasmid with NdeI and BamHI double digest, which doesn't go to completion, but I gel extract and pool 5 digestion reactions.
I then also double digest the PCR product (I have also tried sequential digests) and purify it with qiagen kit.
In my ligation, i put few varying ratios of insert vs. vector and do an overnight 16C ligation. I heat inactivate the ligase and use 5 uL of the reactions for transformations into BL21 electrocompetent cells. The transformation efficiency is good as my control (the initial plasmid) seems to produce a near lawn. However, I don't get any colonies for my actual trials.
What could be the problem?
I tried to pinpoint the problem by running my ligations on a gel but I guess you can't really judge anything from that..
Really would love some help. Thanks for your time.
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Old 10-15-2011, 07:45 PM
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Default Re: Yet another cloning problem

Quote:
Originally Posted by kbta View Post
So I have a plasmid that contains an insert with NdeI and BamHI into pet3a. My aim is to do an N-terminal deletion. So I have designed a Forward primer with NdeI site and a reverse primer with BamHI site. The BamHI has AAAAAA as extra and NdeI has AAAA as extra. The two have good compatibility and the PCR reaction goes well, where I get the right sized PCR product as my insert.
To get my vector, I cut the original plasmid with NdeI and BamHI double digest, which doesn't go to completion, but I gel extract and pool 5 digestion reactions.
I then also double digest the PCR product (I have also tried sequential digests) and purify it with qiagen kit.
In my ligation, i put few varying ratios of insert vs. vector and do an overnight 16C ligation. I heat inactivate the ligase and use 5 uL of the reactions for transformations into BL21 electrocompetent cells. The transformation efficiency is good as my control (the initial plasmid) seems to produce a near lawn. However, I don't get any colonies for my actual trials.
What could be the problem?
I tried to pinpoint the problem by running my ligations on a gel but I guess you can't really judge anything from that..
Really would love some help. Thanks for your time.

5ul of a ligation seems like way too much to use for electro-competent cells. I use 0.5 - 1.0 ul with chemically competent cells and they are much less efficient than electrocompetent cells. How many nanograms of plasmid are you using for your ligation reaction? Most protocols for electrocompetent cells involve diluting the ligation reaction and then using a small amount of the dilution. This is because the salt in the ligation increases the conductivity of the buffer and can fry your E coli.

Also I would not transform ligations into BL21 cells. BL21 are not a cloning strain and have much higher plasmid mutation rates than standard cloning strains like DH5-alpha. I would transform your ligations into DH5-alpha, then do minipreps and identify correct clones by restriction digest and sequencing (this is essential because your insert is a PCR product and could therefore contain mutations). These could then be transformed into BL21 for your protein expression experiment. I know some people keep -80C BL21 stocks transformed with plasmid, but it is recommended to re-transform fresh each time. Also even using an IPTG inducible system there can be leaky expression of toxic proteins in BL21 that makes it difficult to recover frozen stocks.

My advice would be to try the cloning with a standard cloning strain (DH5-alpha) and see if you still have a problem. I recommend always doing a minus insert ligation control if you aren't already doing it. If you still get no colonies on your +insert and -insert ligations I would suspect that there is something wrong with the ends of the insert.

The more common problem is that there are lots of colonies on the -insert plate indicating the vector is not completely double-digested.
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Old 10-16-2011, 07:13 AM
Pipette Filler
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Default Re: Yet another cloning problem

Thank you mmorgan for your reply.

The salt in the ligation mixture was really messing with my transformation efficiency for sure. I just transformed another set of BL21 (before your reply) with smaller doses of the ligation mixture and the electroporation machine seems to actually finish the 5ms instead of finishing at 2-3 ms. On this subject, is it worth doing purifications on ligation reactions before transformation?

I was not aware of the BL-21 problem. These were just the electrocompetent cells in the -80C. I will try borrowing DH5alphas and cloning into them tomorrow.

I am not quite sure what you mean by a -insert ligation. Is that just ligation with the cut vector? In this case, since I'm using 2 enzymes to cut the vector, they won't ligate. Am I understanding you wrong?

I had the incomplete vector digestion problem until I did a gel extraction. As the gene I cut out from the plasmid to make the vector is about 700bp, I can easily select the double cut enzyme. Now that I've done it, I don't get the false positives on the plate.
Some people have issues with gel purified vectors for ligation, but most say one should gel purify, what are the bases for these 2 opinions?
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Old 10-16-2011, 12:51 PM
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Default Re: Yet another cloning problem

Quote:
Originally Posted by kbta View Post
Thank you mmorgan for your reply.

I am not quite sure what you mean by a -insert ligation. Is that just ligation with the cut vector? In this case, since I'm using 2 enzymes to cut the vector, they won't ligate. Am I understanding you wrong?

I had the incomplete vector digestion problem until I did a gel extraction. As the gene I cut out from the plasmid to make the vector is about 700bp, I can easily select the double cut enzyme. Now that I've done it, I don't get the false positives on the plate.
Some people have issues with gel purified vectors for ligation, but most say one should gel purify, what are the bases for these 2 opinions?

The -insert is to make sure that your vector is actually completely double digested. You're correct that it should not re-ligate and that's exactly what the control is meant to test. If you're cutting out an insert to prepare the vector, this is less of a problem since you can monitor the digestion by appearance of the 700bp band. It's more important when you can't see the difference between single cut and double cut on a gel.

I always gel purify my vectors. Usually I do double digest of 1-3ug DNA for 3-4 hours then run on a gel and purify with QIAEXII glass beads. I usually use 10-50 ng of vector for ligations using Epicentre "Fast-Link" ligase. This enzyme works in 5 minutes at room temperature so its handy if you're doing lots of cloning. I think it's actually cheaper than NEB ligase but don't quote me on that.
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