T/A cloning of PCR products
melansond at am.abru.cg.com
Thu Nov 21 13:46:20 EST 1996
***I'm going to give T/A cloning a try but I've run into a choice problem.
***I've found several kits in the catalogues and now I have to decide. I
***like to "shop" at Invitrogen (their kit is $260.00 for 20 reactions) but
***the Promega catalogue says theirs is only $20.00 for the same number of
***reactions. Typo? Bargain? Any and all experience you can relate
***with this cloning technique will be helpful and appreciated.
***John MacDougall, Ph.D.
***Dept. of Cell Biology
If you are refering to Promega's pGEM-T vector, I can tell you my experiences.
I was working with the TA kit from invitrogen at my precious school, and had
fair success with this vector. The cloning went fine, and I was able to
sequence with no problems. The vector gives adequate copy number in straight
forward minipreps. I also really appreciated the EcoRI sites which flank the
insert, and the unusual NotI site.
As for the pGEM-T, The cloning went fine (no problem, and you can really stetch
the vector to 200 reactions if need be,[I have]). However, the multiple
cloning site leaves a bit to be desired, No EcoRI, and some difficult enzymes
in double digests. And in my hands, the copy number varies from culture to
culture, so I have resorted to re-PCR amplifying the insert out of the vector
instead of digesting.
If I were to buy another kit, I would probably go back to Invotrogen, just for
the MCS. But, I have managed fine with pGEM-T, and I have stretched the vector
well beyond 20 reactions.
My suggestion to you would be to take some time to study the MCS and determine
which sites will be best for you, and your needs. As far as cost is concerned,
there really is no way around it as you cannot buy the vector alone. However,
it is probably worth the price not to have to make your own.
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