I am having an issue trying to clone 1.5 and 2Kb fragments into a lentiviral vector. I successfully cloned a different insert (1Kb) into the same vector on the same day. There were a lot of colonies on the blank (cut vector + ligase with no insert), but about half of the ligated colonies were correct. My problem inserts were cut and ligated at the same time, but came out of the incubator with no colonies (none on the blank either). (A different intact plasmid was also transformed at the same time and looked fine with lots of colonies.)
The only differences between the ligations was one restriction enzyme I used (PspXI + EcoRI worked, but PspXI + NotI did not work), and the DNA itself.
Does anyone have any thoughts? I got advice that some restriction enzymes "over cut" and reducing digest time helps sometimes, but reducing digest time to 5 min did not give colonies either.
Did you check that those combinations of enzymes are compatible with the same restriction digest buffer?
NEB has a great online tool for how to do double-digests. Here's the recommended protocol from the site
Notes:
https://nebcloner.neb.com/#!/redigest
I assume you did a column cleanup (can't heat-inactivate the PspXI). And that you phosphatase-treated the vector so it is less likely to self-ligate.
It's also possible for enzymes to expire/degrade. Check the expiration date on the tube for your NotI.
Good luck!
Thank you Katie A S Burnette for taking the time to answer!
I did use the correct buffer (it was NotI-HF I was using) and I purified the fragment on a gel. Let me know if you think that would contribute somehow to one set of enzymes but not another.
I did not phosphatase treat the vector, but the most pressing issue I'm having with the NotI cut is not related to this, because I don't see any colonies on the blank. This would be the same for if the enzyme was no good; we would expect to see colonies on the blank.
Let me know if I'm off base with any of my assumptions!
A gel clean-up means that you will lose a LOT of your fragment during the cleanup process. Which can be OK if you start with a lot of template molecules.
You need to phosphatase treat the vector. Otherwise it will self-ligate at a much higher rate than ligating in the insert.
If you got 0 colonies, then the issue might be with the transformation or with that tube of competent cells and not with the enzyme. Sometimes you just have to try again. Set up several tubes for that vector + insert. Spread out the transformed cells on several plates of selective media at a few volumes.
Are you using chemi-competent cells or electro-competent? If it's electro and you get any sort of arc/spark/pop, then the cells got killed in the cuvette. If chemi, then you have to be very careful of the temperatures & time.
For both types, let the cells thaw completely on ice & once a tube is thawed you have to use it or throw it away.
Good luck!
Thank you Katie A S Burnette !
I understand your point about phosphatase treatment and will consider it in the future if that becomes an issue, but I'm looking for the reason I'm getting no colonies on blank and ligation only when I use a specific enzyme combination. The other ligations I did on the same day turned out fine, so there is not a general issue with the transformation. I tried this a second time and got the same result.
We are using chemi competent cells and thawing fresh aliquots for single use.
Let me know if there is anything else you can think of that would explain this!
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