Q: Trouble with Kindergarden Ligation (SspBI/ApaI)

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Tue Sep 23 02:17:07 EST 1997


Wolfgang Schechinger (wgschech at med.uni-tuebingen.de) wrote:
> I simply can't insert a sticky 
> fragment: I'm using two primers (one carries the mutation) to 
> generate the PCR fragment. Each primer has a 
> restriction site at one end (SspBI and ApaI), flanked by four 
> additional bases (...GGGCC|C-TATT (5' overhang) for the ApaI site, 
> GAGC-T|GTACA... (3' overhang) for the SspBI site).

Try the following workaround:

- polish the ends of your PCR fragment with Klenow + dNTPs (might not
  be necessary since you use a polymerase with exonuclease activity)
- clone insert into a pBluescript cut with EcoRV
- pop out your fragment with the two restriction enzymes you want to
  use

This worked for me one time when I ran into the same problem as you.
I have no idea *why* it is easier to subclone a plasmid fragment
than to subclone a PCR fragment, but here we go.

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP3 */
/* "Science is the game we play with God to find out what His rules are."  */



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