Subcloning PCR

Bernard Heymann bheymann at
Sat Sep 3 12:04:54 EST 1994

In article <199409020012.RAA17831 at>,
jloring at ("Jeanne Loring") wrote:

> more carefully.  I cut my fragment with the restriction enzymes whose sequence I
> believed I had included (with a 5'clamp), but there was no way to be sure they
> both worked (SacI and XhoI).  Now I'm not getting inserts into Bluescript II.  

You should look at the NEB catalogue, p. 180, where they give a very useful
table of the restriction efficiency of cassettes of different length by
various commonly used enzymes. Sac I seems to require more than one extra
base pair, and Xho I at least three more flanking the six-bp recognition
sequence. I find it a good practice to design primers of 20 bases or more
with the restriction site in the middle - that way you are sure it is not
too short to be cut. The other things I do is to purify the fragment away
from the Taq (which can screw up your sticky ends), and digest the purified
fragment overnight (shorter times have given me much lower or no yields).

Good luck

Bernard Heymann
bheymann at

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