I insert a sequence into the plasmid at the PspLI and BamHI restriction sites. The sticky ends of these restriction enzymes share two of four identical nucleotides. This likely explains why I get only clones containing plasmids without inserts. I will perform additional ligations, increasing the insert-to-vector molar ratio to 5:1 or 7:1. Are there other methods to favor insert integration? Perhaps adding a small amount of formamide? I would appreciate any advice.
Agree with Paul on treating the vector with alkaline phosphatase to help prevents self-ligation. You could also consider adding ligase enhancers like PEG to promote macromolecular crowding and increases ligation efficiency. Just bear in mind that PEG concentrations above 5% and extended incubation with PEG can reduce transformation efficiency. You might want to try run some trials for the optimized condition.
You should also consider incubating at the recommended temperature (in your protocol) overnight. Adding phosphate buffer and a longer incubation time should work, from experience.
Wish you luck.
Dear Bimpe Suliyat Azeez and Annie Huang,
Thank you very much for your valuable advice. I believe I can handle this insertion now; you have provided me with many ways to solve the problem.
Looking at PspL1 (C/GTACG) and BamHI (G/GATCC) I don't think your problem is compatible sticky ends but rather poorly digested DNA (maybe the two sites are too close to each other)
Dear Didier,
I am attaching a picture of my restriction sequence. As you can see, the distance between the sites is sufficient, and there is complementarity on two letters. The restriction enzymes appear to be working well, judging by the agarose gel. Thank you
I have never seen such a fusion between two incompatible restriction sites .1/ did you sequence your clones to see the fusion site? or 2/did you tryed a Sal1 digestion; if you are right the SalI site should disapear... (and the bamHI too...
if such fusion was possible then molecular biologist would have been in big trouble since a long time...
Dear Didier,
I did not sequence after ligation due to limited capabilities, reserving sequencing for critical checks. I successfully inserted into this vector twice. However, with the third insertion (PspLI-BamHI), I encountered the problem: PCR analysis of several dozen clones revealed empty vector. Both restriction enzymes are functional, indicating highly effective vector self-ligation. This is likely due to partially complementary sticky ends.
I plan to dephosphorylate the vector next week. If the insertion is successful, that is good. If there is still no result for PspLI-BamHI, I will try PspLI-SalI.
sorry but I was suggesting to do a Sal1 (or BamHI) restriction analysis on the clones minipreps you obtained ...
Dear Didier, thanks for the advice. I have an isolated plasmid from one empty clone, so it is not difficult for me to restrict it with SalI. If it does not work, there is a high probability of self-ligation by PspL-BamH.
I have assembled the plasmid, which was time-consuming. Initially, I treated the cut vector with phosphatase. The number of clones after ligation decreased, and the remaining ones lacked the insert. Following Didier Poncet's advice, I analyzed one of the null plasmids by restriction. All restriction sites (SalI, PspLI, and BamHI) and fragment sizes were preserved. The plasmid was identical to the original one into which I attempted to insert. This means that Didier was right: the problem was not the similarity of the PspLI and BamHI restriction sites. During one of the phoreses, I observed that PspLI did not completely digest the vector, despite its normal performance during testing. Given that I use a significant amount of the original vector for plasmid assembly, and distinguishing between single and double-digested vectors is challenging with simultaneous restriction by PspLI and BamHI on phoresis, I hypothesized that the issue stemmed from incomplete or unstable PspLI activity. The presence of an single-digested vector outcompetes the double-digested one. I purchased a buffer from the PspLI manufacturer, which ensures 100% activity for both PspLI and BamHI (the one I used previously yielded up to 75%), and added BSA. This resolved the issue. Thank you to everyone for the advice.
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