Hi all,
I have an insert that I am attempting to ligate into pcDNA3.0 cut with HindIII and KpnI. I get a ton of background colonies even after dephosphorylating the vector (I know my phosphatase treatments work). I realized that HindIII and KpnI are right next to each other on pcDNA3.0. Therefore, the problem might be that either one or both enzymes cannot cut (sterics). Would performing single digestions followed by purification for each enzyme be the way to go? I could change my sites but I would rather not.
Any suggestions?
Hi there,
Sequential digest is definitely a good option to avoid competition between enzymes (if you have a system where both enzymes are compatible in the same buffer run the first digest then inactivate the first enzyme by heat treatment and then add the second enzyme. So there would be no purification required between digests).
To get a clearer picture of the situation run ligation controls in parallel with your cloning. These controls are:
ligation mix contaning only the digested vector (no insert no ligase)
ligation containing digested vector and ligase (no insert).
The first will tell you about the presence of intact plasmids in your vector prep and the second will tell you about the self ligation event (if you get more clones than with the first control).
Hi Dominique,
Thank you for the response. When I mention background I am referring to the second control you proposed.
Thanks again.
I agree that the proximity of the two enzymes is causing the problem. One enzyme is cutting fine whilst the other one doesn't cut at all due to a too short overhang i presume. Try to combine different combinations with digests i.e. first with KpnI, heat activate and then with HindIII for example. This helped me quite a lot in the past. Also lowering the concentration might help. For enzymes from Thermo scientific, use a unique buffer like BamHI for this single/double digest.
Good luck!
Hi again,
So you a a high level of selfligation and/or of undigested plasmid. Perform the first control to get sure : if no clones, it's a selfligation problem and if clones it is also a problem of digest efficiency.
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