I am using a plasmid vector of about 8kb for cloning. After cutting with EcoRI and BamHI, I performed a ligation with T4 DNA ligase and transformed in DH5-alpha, and found that there were as many colonies in [double cut vector + insert + ligase] group as in my [double cut vector + ligase], [single cut vector + ligase] and [uncut vector] controls (in 1000s). Transforming with the double-cut vector alone gives few 10s of colonies.
Doubting the restriction enzymes, I cut the plasmid individually with both enzymes and found by electrophoresis that they linearise the plasmid. I tried a simultaneous digestion with both enzymes (mutually compatible buffer), and also tried sequential digestion with BamHI followed by EcoRI and vice versa, with cleaning up the first digest using a spin column or phenol-chloroform extraction after heat inactivation. The problem persists.
Hi Devi,
The large number of colonies you are seeing is most likely due to uncut vector/single cut vector in your ligation. There are always going to be a small amount of these molecules left after double-digestion. One way to reduce the number of these type of molecules is to digest overnight and use 5X amount of enzyme to vector (5 units enzyme/1ug DNA). Additionally you should agarose gel purify the linear vector away from any uncut vector. Just because you can't see any uncut vector in a gel doesn't mean its not there. It may be below the limit of visualization (
Hi Devi,
it sounds as if you have already covered the most obvious causes.
One question I have is how close to each other the restriction sites are? A quick check on NEB site shows that BamHI requires 3bp and EcoRI 5bp extra at the end for good cleavage.
On a practical level (especially if the sites are close) try treating with alkaline phosphatase, to stop the re-circularisation of the single cut vector. It will reduce the efficiency of cloning a bit, but the removal of the background helps find the few that have an insert.
-Richard
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
Hi Tom,
I am using fast digest enzymes which have incubations of 5-15 mins. EcoRI shows star activity after 30 min and BamHI after 1 hour.
As I had mentioned, I did have a control with double digested vector without ligase. That gave me about 20-50 colonies in different trials. And the double digested vector with ligase gave a full plate of colonies. Since the single cut vector seems to be way more than the uncut, I was trying to troubleshoot that. Also, running the uncut and linearised vector side by side on a gel shows that the linearised form is very close to one of the forms (probably supercoiled) of the uncut. Even after running long on a 0.7% gel, I could not seem to resolve them apart. So I refrained from gel extraction. Do you think I should still go ahead and try it out?
Hi Richard,
The sites are 25 bp apart on the vector. Alkaline phosphatase has been on my to-do-list. I was about to set up the reaction tonight. I'll update on what happens.
Hi Devi,
20-50 colonies on the cut vector with no ligase control is not good so yes I would still recommend doing a gel extraction. Anything you can do to reduce the background colonies is worth it. Seems that your problem is a lot of single cut vector due to the fast digest enzymes. You may want to try using normal (non-fast digest enzymes) if you can and run the digests overnight, gel purify and proceed from there.
I would as well suggest a dephosphorelation of the digested vector (we use Shrimp alkaline phosphatase) followed by gel electrophoresis and purification from the agarose gel. In empty Vector ligation controls I only have few to non colonies.
Hi all,
Thanks for the suggestions. I did an alkaline phosphatase treatment and gel extracted the DNA. Surprisingly I got two bands on the gel, which previously gave me only one. Then performed a ligation. Now I have abut 30+ colonies in only vector, 50+ colonies in vector + ligase and 300+ colonies in vector + insert + ligase. So it worked. I just might have to screen more colonies. But I still do not understand how I had so much of single cut species in the first place.
Hi Floris,
That's a great suggestion. Unfortunately the site between the two sites is present 3 times in my insert. As of now I managed to get a positive clone. I'll try out your idea in future.
Hi Devi,
i have a problem like th one you mentioned here..i do the electroporation with the self ligated vector and double digest vector, and do the electroporation seperately for them. i repeat this 3 times and all the results were same..few colonies in doube digested and no colonies for the self ligated vector.. its understandble to say that there may remain some uncut vector in the double digested one which give rise to the colonies, but how about the self ligated one? why there is not even a single colony?
note: this is done to check the vector and there is no insert.
note: we did the self ligation for the double digested vector with T4 ligase.
Thank you
Hi Samira,
What do you mean by "self-ligated vector"? Was it cut by a single enzyme or two enzymes? After ligation, do you clean the DNA using phenol-chloroform or a spin-column? Or just heat-inactivate/directly electroporate?
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