PCR using d(T) 19mer

maize at bragg.bio.purdue.edu maize at bragg.bio.purdue.edu
Sat Jan 2 15:15:03 EST 1993


  I'm trying to do anchored PCR using only a dT 19mer and nested specific 
primers.  But (probably not surprisingly) dT19 is a cruddy PCR primer.  At 
42 oC annealing temp, I get no amplification, at 37 oC I get a smear at 
high molecular weights (like, above 1.6 kb.) and subsequent reamplification
with the nested primer gives me the same thing.  (The real band should be 
less than 600 bases.)  MgCl2 concentrations between zero and 9 mM have been 
tried.
  I'm trying to calibrate the rxns by using a previously cloned cDNA.  I 
use 5 pg in a rxn.  Amplification using either of the specific nested 
primers and a sequencing primer (the universal primer from the vector) gives 
me a clean band at the right size -- the oligo dT 19mer gives me the 
results I mention above.
  Any suggestions?  All accounts of anchored PCR I'm aware of either avoid 
using an oligo dT mer or don't mention any difficulty in using it (other 
than suggesting the use of nested internal primers.)
  Please email me, if possible, I'm out of town for a week and may not be 
able to read a posted response before it expires.

--Phillip
_______________________________________________________________________
Phillip SanMiguel               Purdue
maize at bilbo.bio.purdue.edu       Institute for
                                  Molecular    "We'll do anything
  Bennetzen Lab                    Plant          for funding."
                                    Science
_______________________________________________________________________



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