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

[David J. Meyer]

David J. Meyer (ez005587 at chip.ucdavis.edu) wrote:
: 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.

Sorry for not including this info first:

1) I would like to use total RNA from plants, probably contaminated with
carbohydrates, as the starting material

2) Primers are 25-30mers, Tm's ~55 degrees C in PCR

3) Targets are maximally 1.1 kB

4) Is a gene-specific primer for the RT step better/necessary with rTth?

5) (anything else I have forgotten to mention?)

Hope to hear replies soon!

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: ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
: David J. Meyer
: djmeyer at ucdavis.edu

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David J. Meyer
djmeyer at ucdavis.edu