LA PCR

POPOVA ANDREI.POPOV at afrc.ac.uk
Sat Sep 24 08:57:55 EST 1994


DEAR COLLEAGUE,

IT IS NOT EASY TO GIVE ANY ADVICES ON LONG PCR AFTER
THE INTRODUCTION OF "BARNESIAN" METHOD (SUMMARY- TIBS,1994, 19:342),
HOWEVER, WITHOUT USING SOFISTICATED POLYMERASES MIXTURES
YOU CAN TRY:
1 INCREASE THE pH OF TRIS BUFFER TO 8.8-9.2
2 CALCULATE THE EXACT TIME NEEDED FOR TAQ POLYMERASE TO EXTEND
  YOUR FRAGMENT AT 72 C (AVERAGE PROCESSIVITY OF THIS ENZYME IS
  3000 BP/MIN) AND DO NOT EXEED THIS TIME BY MORE THAN 20-30%.
  I HAVE AMPLIFIED 2.5 KB GENOMIC FRAGMENT AT 1 MIN EXTENSION 
  TIME AND COULD NOT DO IT BEFORE USING 2 MIN EXTENSION TIME.
  IF OBSERVATIONS OF W.BARNES ARE 100% TRUE, IT COULD BE THAT
  DURING LONGER EXTENSION TIMES UNMODIFIED TAQ POL SIMPLY DESTROY 
  YOUR PRIMERS, HOWEVER, ONE SHOULD BE ABLE TO CORRECT THIS PROBLEM BY SIMPLY
  ADDING FRESH PRIMER EVERY 5 CYCLES.
3 TITRATE MgCl2- SOMETIMES YOU GET WONDERFUL AMPLIFICATION BY
  JUST CHANGING MgCl2 CONCENTRATION BY 0.5 mM
4 IF YOU GET A SMEAR OF SUSPICIOUS MW YOU CAN TRY NESTED/SEMI-NESTED PRIMERS
5 HOT START IS A MUST
6 MELTING CYCLE SHOULD BE LONGER THAN 30 SEC
7 TEMPLATE SHOULD BE REALLY CLEAN

GOOD LUCK
ANDREI POPOV





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