5 replies to this topic

[RichardColes]

Hi I hope someone can help. I am trying to clone a murine TCR, and to do this i have introduced a ClaI and BglII site into the PCR product. I have done this and cloned it into pGEMT, and sequenced it to check that the sites are there, they are. Also there is a unique BamHI site in the insert as well. Here is the problem. I try to cut the vector with the above three enzymes and also XmnI (unique to pGEMT) and I get what looks like partial cutting (but i dont think it is) with all four enzymes. If however i cut a control plasmid with the XmnI enzyme without the insert the cut is complete.
1. I have tried to purify the mini preps so they are clean.
2. I didnt overload the system with DNA so that some would be uncut.
3. Sequence looks fine.

Q: Why does the pGEMT specific site cut just as bad as the others when a control plasmid prepared the same way cuts well???
Q: Can the DNA insert create some weird secondary structure that makes the plasmid hard to cut??

I urgently need help with this because i have been held up at this point for over 12 months. I have tried so many things, so why isn't it working??

Thanks for your advice guys.

Rich.

[Maitreyis]

hi! i am facing a similar problem. well, i suggest you do a double digest of your construct, that is, digest with enzymes such that one cuts the insert while the other cuts the vector. this should liberate a fragment. if that is not the case then u probably have some contamination in your vector preparation.  also when you transform your ligation mixture, analyse some non recombinants. these should behave like your vector. if not then just start all over again with plasmid prep of vector from a single colony. this should help to avoid any external contamination.
i hope this helps you.
All the best.

[RichardColes]

Thanks for the advice. I did another digest with two enzymes both with in the pGEMT and got a fragment however a lot remianed uncut so it seems as if the vector can be cut but that only about a 1/3 of it actually is cut the rest remains uncut. A bit bizzare but if anyne else has sugestions as to what i might do to get it to fully cut, post away!!

Thanks.

[Maitreyis]

Are you sure that your strain is fine? I will once again stress on the analysing non recombinants which you will get along with your recombinants on transforming the ligation mixture. These recombinants should behave like the original plasmid. if not then u are sure that u have a contamination. this simple thing sure helped me stop a fools chase!!

[RichardColes]

I did do a blue colony (no insert) and it behaved fine. It was able to be cut 100 % , unlike the ones without the insert.

[Svengali]

Cla I is dam methylation sensitive.  Could it be that the Cla 1 site is being methylated by the bug you are transforming it into?  If it is, you will need to transform it into an methylase deficient E.coli strain.
This is a real possibility by the way, i have experienced it.