what's the best way to clone PCR product

POPOVA ANDREI.POPOV at afrc.ac.uk
Sat Oct 1 11:12:16 EST 1994


 I AM LITTLE  BIT SURPRISED THAT NOBODY
HAS YET MENTIONED THE METHOD PUBLISHED IN NAR (1993).

BREIFLY,  THE PRIMER IS DESINED WITH SHORT RE
SEQUENCE THAT CAN BE REDUCED TO CLONABLE END BY KLENOW FRAGMENT.
FOR ECO RI THIS SEQUENCE WILL BE:

       5'-AATTCNNNNNNNNN-3'

AFTER AMPLIFICATION YOU GET:

       5'-AATTCNNNNNNNNNNNNNNNGAATT-3'
       3'-TTAAGNNNNNNNNNNNNNNNCTTAA-5'

AFTER KLENOW (IN THE PRESENCE OF dGTP, dCTP):

       5'-AATTCNNNNNNNNNNNNNNNG     -3'
       3'-    GNNNNNNNNNNNNNNNCTTAA -5'

THE SAME APPROACH CAN BE USED WITH APPR. 20-30 OTHER
RESTRICTION ENZIMES, YOUR PRIMERS
WILL CONTAIN LESS OF MISPRIMING NUCLEOTIDES, AND BE A LITTLE
BIT CHEAPER...
AND MOST EXCITING IS THAT IT WORKS.



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