I have been trying to subclone an insert having 2300 bp into pGEm T easy vector. The primers I used have SnaBl and Notl site in forward and reverse primer end respectively. In my knowledge, digestion of the PCR fragment with SnaBl and Notl before ligating them directly to the vector might not be a prudent idea as restriction sites are very close to Primer end,especially for Notl I am little worried. I decided to use pGEM T easy for doing TA cloning before I obtain a properly digested PCR product. Unfortunately, I did not get any insert. Is the insert size too big for a pGEM T easy vector? I used JM109 competent cells for transformation.Obviously, this is my First try. If so, how can i get rid of that problem?
Hi!
I did cloning 7 kb insert amplified from animal genomic DNA to pGEM T easy vector and got many colonies with correct insert. I used electro-transformation and DH10B cell.
If you had colonies but without insert, make sure your polymerase produce A-overhang products.
Did you use fast ligation protocol? If yes, try to ligate overnight at 8-16C and take as much of insertion and possible.
I did not use fast ligation protocol but yes, I was in rush. I incubated at room temp for 2 hours following overnight incubation at 4 degree
Hi all, I used 25 ng of pGEM-T-easy vector and 7:1 insert to vector ration for ligation? Is 25 g too low? I got positive clones before working with 25 ng Vector, but my insert sizes were 800, 900 and 1300 bp . As per your advice, I used DH5alpha com. cell.
hi,
this sounds already way to much insert and backbone for the ligation. In addition the have almost the same fragment size so you should go for rahter equimolar amounts each. Further wouldn't it be easier to just expand you primer sequence and cut the PCR product instead of going this additional way? In this way you could also use a proofreading enzyme as the PCR with a normal taq polymerase will add a point mutation in ~20% of you PCR products when you go for 30 cycles.
Sven
I agree with Sven Reister,
An equimolar ratio is advisable, with emphasis on equimolar and not mass.
Your ligation reaction volume shouldn't exceed 20ul much.
If you'd use primers with non-complementary tails that harbor your restriction enzyme sites directly after the complementary part and about 5-8bp random sequence afterwards, you can use a Q5 polymerase (e.g. NEB, M0491S). This can get you an error-free PCR fragment in 25 cycles, that you then may clean up, double digest, clean up and use for the equally double digested and cleaned up vector in a ligation.
I recommend up to ten 50ul PCR reactions for the same insert, as to have a good amount of DNA for all the cleanups (you can pool them).
4-16°C overnight ligations work best for me, with subsequent DH5-alpha (>10^8 cfu) electroporation.
Kindest regards
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