RT PCR-any suggestions?

[Too Heng-Phon]

Tracy Aquilla (aquilla at salus.med.uvm.edu) wrote:
: Hi World,
:     I am thinking about using RT PCR to clone several cDNAs, using total RNA
: as template. I am wondering if anyone who has used this technique has any
: suggestions for me. Also, I have heard about a new enzyme sold by
: Perkin-Elmer Applied BioSystems division which has both reverse
: transcriptase activity as well as DNA polymerase activity. I think this
: sounds like a good thing, but again, I would like to hear from anyone who
: has used this enzyme. Any suggestions are welcome. Thanks for the help!
:     Tracy
: Tracy Aquilla, Post-doctoral Research Associate
: Department of Molecular Physiology and Biophysics
: University of Vermont, College of Medicine
: aquilla at salus.med.uvm.edu

     Dear tracy,
               RT PCR is a doodle and is simple like hell.  The beauty is 
that it does work!!  Here it is:  4 ul total RNA + 19 ul of H20, 1 ul 
random primer (or oligodT) then heat to 95 C for 5 min.  Chill it by 
placing on ice (or the freezer) for about 5 min.  Then add 2 ul of RNAse 
inhibitor (100 U), 8 ul 5X RT buffer(e.g., Promega, contain DDT), 4 ul 
dNTP (10 mM of each nucleotide), 2 ul MoMulV (400 U total).  Then 
incubate at 42 C for about 1 hr or more.  Then, 2-5 ul for PCR (depending 
on the abundance of your transcript).
               Comment:  success depends alot on the quality of your 
total RNA.  Make good RNA (if you need help, shout..we'll help ya).  
Promega stuffs are useable and cheap.  More expensive gimmicks are like: 
superscript from BRL.  MoMulv or AMV can be used.  Yet to try rth (perkin 
elmer), ask again next few weeks, may have some info for you.  Best of 
luck.              Phon