Hi everyone,
I am currently trying to clone a fragment (~1700bp) cut with ECORI into a vector (~5300 bp) linearized by ECORV. Since ECORI leaves cohesive ends I am using Klenow Fragment (Polymerase I large fragment from NEB) to fill in the cohesive ends and make them blunt. This is the detailed procedure that I am following:
- digest vector with ECORV and fragment with ECORI overnight at 37 in cutsmart buffer (NEB)
- add 1uL CIP (NEB) to linearised vector and incubate at 37 15 minutes.
- add 1uL 10 mM dNTP mix and 1U (0.2 uL) of Klenow to fragment and icubate at 25 for 15 minutes.
-Then I run everything on a gel to purify bands and perform ligation.
-Ligation is done using T4 ligase, 150 ng of fragment and 50 ng dephosphorylated vector, incubated room temperature for 2 hours.
A negative control with 50ng vector only is done to check CIP efficiency.
-Then cells are transformed, plated and incubated at 37 overnight.
The next day I checked the plates and I saw colonies in both, the colonies on the negative control (dephosphorylated vector only) were more or less half in number compared to the ones with vector + insert. Then I picked 30 colonies from this plate and I did a colony PCR to see which one had the insert inside. All of them resulted negative. I am afraid it is not only bad luck, but something went wrong with my cloning. I think something was wrong with the Klenow enzyme, maybe the fact that I did not inactivate it. Do you have some ideas on what might have gone wrong? if you did something similar to what I need to do, do you have some suggestions? If you need more details I am more than happy to give them to you.
Thank you very much
Ali Mahmoudpour thank you very much for your answer. I am already cutting my fragment with ECORI but I cannot cut the backbone with the same enzyme because it has 2 sites for ECORI.
Ali Mahmoudpour thank you. the backbone is a plasmid called phd_dsRED in which I already inserted an insert using cohesive cloning with ECORI, that is why I cannot use a second time ECORI. Yes I am aware about the difficulties, what is weird to me is that the number of colonies on my plates is promising, but when I check them they appear to have something wrong inside.
Hi, I agree with Ali, blunt end ligation is quite cumbersome. I'd also suggest to increase the amount of fragment higher than 3:1 ratio (like 5:1) and maybe also spin columns for purification. Last (or first), maybe overnight incubation is a bit too long for enzymes with star activity like yours, try with shorter times.
If the reaction still does not work, you might consider adding a tail to your fragment (pcr) with a site for a restriction enzyme that may be suitable foryour vector. Good luck!
Hi Lorenzo,
As far as I undedstand you see a relatively large number of colonies in your cip ctrl.
I suggest you to:
-1) Add another control: digest your pladmid with eco then transform with no ligation. This tells you if the colonies in the control plate originate from a limited efficiency of your eco enzyme or from a limited efficiency of your cip.
2) Avoid the agar purification and use columns, as uv may damage the ends of your dna, hence reducing the overall efficiency.
3) add a positive control (eg ecor1 ecor1 ligation) to check your ligase
4) increase the fragment/plasmid ratio up to 10:1
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