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[jen123]

I am trying to create a library by directional cloning of a 1.8kb PCR product into a 5.3kb vector. I use a previously prepared 7.1kb construct (derived from pET21) as a template in a PCR reaction to amplify a 1.8kb insert band. I digest the same 7.1 kb construct with Not I and Nde I (which effectively removes the 1.8 kb band) and purified the 5.3kb fragment which I use as my vector. I also digest the insert which is my PCR product with the same enzymes. I made sure that my primers contained >6 bases outside of the restriction enzyme sites to ensure efficient restriction digestion of my PCR product. After ligation and electroporation there are no colonies on my plates. I also transformed with my original uncut 7.1kb construct to check the transformation efficiency of my competent cells but that is not the problem. I ran 4ml of my completed ligation reaction on a gel and observed a ladder of bands. I gel purified some of the bands, re-cut with Not I and Nde I and ran this on a gel and got back bands that corresponded to my original vector and insert. Hence ligation is occuring between insert and vector but not efficiently.

I was worried that my PCR product does not digest efficiently after all, so I decided to cut my original 7.1kb construct with Not I and Nde I, purify both bands (5.3kb and 1.8kb) and try to re-ligate this. I digested consecutively even though a double digest is possible to make sure that the vector first linearises and then that I get the two bands corresponding to doubly cut vector. Since after digestion I see only the two bands of the correct sizes it is safe to assume that digestion was complete for both the "insert" and the "vector". After ligation overnight in a total volume of 15ml at 16°C. I ran 4ml of my ligation on a gel and once again noticed a ladder of bands. I wanted to determine whether any of these bands are circularised to I digested one of my ligations with an exonuclease. I found that the most of the ladder disappeared meaning that my ligation consisted mainly of linear fragments. I am assuming that since I see this ladder things are ligating together albeit not in the manner I want it to and hence the ligase is working. I then decided to cut my original 7.1 kb construct with Not I and Nde I separately to determine whether single digestion with these enzymes can be ligated. I ligated overnight at 4 and 16°C. Again, I ran the completed ligations on a gel. The constructed digested with Not I gave two bands. However, the construct digested with Nde I gave me a ladder of bands one of which corresponds to my linearised construct. So I assumed that the construct digested with Nde I was not re-ligating efficiently. This made me suspect that the reason my original directional cloning does not work has something to do with inefficient ligation of DNA cut with Nde I. However, when I transformed these ligations I got colonies for the Nde I digest and none for the Not I digest. I also digested my construct in parallel with Pst I and Bgl II to see whether these single digests can re-ligate and transform and they do. I do not understand what is wrong It appears to have something to do with Not I but I cannot think what the problem can be. Has anyone else ever experienced something similar? I am at a loss as to how to proceed. Any helpful advice would be much appreciated.

[5'GCACGTTGGTATAAT]

Hi,

I recently had a similar problem trying to get a 500 bp PCR product into pET43 for expression using PCR introduced SpeI and XhoI sites. I eventually resolved the problem by T:A cloning the PCR into pGEM EasyT and sequencing the pGEM plasmid. Somehow, it apears I couldn't make a SpeI site correctly :S (despite very careful checking of the oligo). Anyway, pGEMEasy T has a few sites which are present in the pET series MCSs, and I just cut at the NotI site and used the resulting NotI x XhoI fragment...successfully.

Anyway, enough of my problems. What I suggest you do is T:A clone the PCR product and check its sequence. Once you know what the sequence is you can decipher what's going on far better. The hidden bonus of this is if you have pGEM Easy T (Promega - other TA systems might also be suitable) you can use a functional vector site upstream to put your insert into pET. In frame and everything, but with a stretch of pGEM sequence sandwiched by pET and insert, if that is acceptable.

HTH - and good luck and I hope you have more patience in solving it than me!