imakcm at leonis.nus.sg (Kuan Chee Mun) wrote:
>I'm currently making some GAL4-cDNA fusion libraries for two-hybrid
>screening. Does anyone know how to control the RT reaction so that it's
>efficient enough to to give decent first strands, and yet not hit the 5'
>UTR ? (that'll mess up the fusion junction with GAL4 on the vector)
>Thanks a 10e6!
>Institute of Molecular Agrobiology
>National University of Singapore
>PS. Any tips to make sure I get all 3 frames in the right 5'-3' orientation?
Welcome to expression library cloning..grin. If you create a
directional library (Oligo dt primed) and take care to make sure that
your final ligation into the vector is the same direction as your
promoter/primer site, the theoretical best you can expect is a 33%
chance that you will be in frame.
As far as the first part of your question I don't know how you'd
interrupt the reverse transcription to only include the translated
portion of your gene...
--Rover The Mad Molecular Biologist
rover at livnet.combarlow at picard.evms.edu