what's the best way to clone PCR product

[Klaus Salger]

POPOVA (ANDREI.POPOV at afrc.ac.uk) wrote:
:  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.

I think T4 DNA polymerase would work even better then Klenow
because of its much higer 3'-5' exonuclease activity.
Cheers
  Klaus


Klaus Salger
Zoologisches Institut
AG MacWilliams
Luisenstr. 14
80333 Muenchen
Germany

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e-mail: salger at zi.biologie.uni-muenchen.de