Need opinions on DDPCR idea

Steven Sullivan sullivan at gwis2.circ.gwu.edu
Sun Jul 9 13:13:33 EST 1995



I do RT-DD-PCR on RNA from a tissue that is exceedingly tedious to harvest
in sufficient amounts for repeated experiments.  I was wondering if the
followign idea was feasible:  do four RTs on my precious RNA using the
four dTVN primers, then do 2nd strand synth and shotgun clone everything
into a suitable vector (e.g. Bluescript), resulting in 'renewable' stocks
of template for DD and PCR -- i.e. I could use the plasmid populations as
DD templates rather than having to go back and collect more RNA when mine
ran out.  My worry is that some RNA's will become lost in the procedure
due to differential ligation efficiency, differential propagation in E
coli, etc.  What do you think? 




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