RT-PCR: which enzyme(s) are best?

Andre Hamel hamel at ccu.umanitoba.ca
Fri Jun 18 09:00:29 EST 1993


In article <C8sFMD.Ao4 at ucdavis.edu> ez005587 at chip.ucdavis.edu (David J. Meyer) writes:
>Hello, netters!
>
>	I need to do some RT-PCR soon, and am planning things now. I have
>previously used MMLV RTase, followed by Taq for the PCR; does anyone have
>any experience/recommendations regarding the use of enzymes such as Cetus'
>rTth polymerase? It would be nice, in theory, to use just the one enzyme.
>Please post replies, as I read this group regularly.
>
>--
>^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>David J. Meyer
>djmeyer at ucdavis.edu


MMLV RT and Taq (Cetus') are a great combination IMHO!

Stick with it! Unless you've definitely got 2ndary structure problems with
your RNA, rTth might be useful, otherwise it's NOT as sensitive as what
you're using.

MMLV RT can withstand use at as high as 50C (more reliably at 45C),
further, RNAaseH minus Superscript (BRL) definitely works okay at 50C,
much better than MMLV RT (we've used NEB & BRL MMLV RTs with no apparent
diff).

regards, Andre
                            ********************
Andre Hamel                                 email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services                lab tel.: (204) 945-7630
Infectious & Genetic Disease                     FAX: (204) 945-8062     
Mol.Biol.Lab., Winnipeg, Manitoba, CANADA
                            ********************



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