I am trying to clone a small gene (180bp) in to a ~6kb vector, using XhoI site only. I've been treating the vector with alkaline phosphatase (Roche). However, I'm getting colonies having self ligated vector. Tried using even 10ng of vector for ligations.
Is there any enzyme which I could use to blunt one end, to avoid self ligation of the vector and increase vector:insert ligation possibilities?
Hi there,
If you still observe self ligation then phosphatase treatment is not 100% efficient. Did you actually check the digest with XhoI was total? If not just run a ligation experiment with the digested vector alone without ligase and compare with what is obtained with the vector alone with ligase. If you get clone from the former some vector hasn't been linearized. If you get more clones from the latter there is self ligation occuring. If not clones are only due to undgested vector.
About your idea of filling one end: both ends are chemically identical therefore thereis no way to distinguish specifically one of the 2.
Hi there,
If you still observe self ligation then phosphatase treatment is not 100% efficient. Did you actually check the digest with XhoI was total? If not just run a ligation experiment with the digested vector alone without ligase and compare with what is obtained with the vector alone with ligase. If you get clone from the former some vector hasn't been linearized. If you get more clones from the latter there is self ligation occuring. If not clones are only due to undgested vector.
About your idea of filling one end: both ends are chemically identical therefore thereis no way to distinguish specifically one of the 2.
Hi Parikh, Your question clearly shows that either vector digestion or phosphatase treatment is not proper. To rule out, as Liger mentioned, you can run digested and undigested vector on an agarose gel and observe if the vector digestion is successful or not. On the other hand, check if enzyme efficiency is hampered.We used to use CIP from NEB especially in very low concentrations, which always worked. Higher concentrations of CIP fails. So check with the concentration of phosphatase you are using and recheck its efficiency.
Hi Kalyani,
the agarose gel experiment might not be conclusive since minute amounts of supercoiled plasmid (not visible on gel) might be sufficient to transfom enough bacteria .
How many colonies are you getting? If it's only a few, you can still get ligation of insert well above your background. If it's much more than that, you have an enzyme problem.
While you're trouble shooting you may want to look at adding a new restriction site to one end of your insert with PCR (I recognize that might not work with your vector).
Thanks a lot everyone for the suggestions.
I get a very clean, single band for the digested vector on the gel, which I gel elute after digestion and AP treatment. However, I'll transform digested vector to see in case the vector is not completely digested/ dephosphorylated. But won't that too give me colonies as the vector is digested by just one enzyme and hence cellular ligases will fill the nicks when the two ends come together in the cell?
My Ph.D. advisor often reminded me to do the experiment to see the answer rather than worrying too much about what could go wrong. If you do get too many colonies, you have an answer. Regardless, it might still be worth your while to borrow or buy a new phosphatase and/or consider a new subcloning strategy.
You can try phosphorylating your insert before ligation with the dephosphorylated linearized vector. This might increase the chances of insert-vector ligation. If there is a time constraint involved, and you don't want to spend too much time troubleshooting this step, like Eric pointed out, if your control plate transformed with linearized vector shows less than 1/4th of the number of colonies you get on your insert ligated samples, you should still be able to easily find a single colony with you desired interest.
Hi Parikh, when you are getting clean, single band for the digested vector on the gel, it means, you vector is either completely digested or its not getting digested atall. Could you run an undigested vector along side? As its a 6kb vector, if you can run both on 1.5% or 2% gel, you will get to know easily if the vector is properly digested or not. Its a good practice to run undigested vector along with digested ones. If you are sure that your XhoI enzyme is working fine, you can work to rule out phosphatase issue too.
Hi Piero, you might be right. I agree its not conclusive. But since its a 6kb vector, the difference between digested and undigested sample can be made out if we can slightly increase the gel percentage (basically an easy approach) though its not conclusive.
As suggested above you need to improved your dephosphorilation activity or either change cloning strtegy. I would suggest to change the restriction enzyme, either in your vector or in your insert, to SalI. they have compatible ends and after ligation the restriction site does not regenerate, then you can cut your ligation with the one that you used in your vector and all selfreligation vector will be cutted again.
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