PCR-cloning

P. Mertens mertens at toxi.ucl.ac.be
Wed Mar 20 07:42:28 EST 1996


I've recently cloned PCR products with EcoR I and Xho I sites at the 
ends; both primers were designed with 3 additional bases at the end. PCR 
products (produced with Pwo) were cleaned with Quiaquick PCR purif. kit, 
then double-restricted with the two restriction enzymes. Three differents 
methods were used to treat restricted PCR products and vector (SK+) 
before cloning:
a. precipitation
b. purif. with Quiaquick PCR purif. kit
c. purif. with Quiaquick Gel extraction kit (after running in agarose 
gel)

All three methods worked. Recovery was the worse with method c, but no 
clones were obtained for the vector alone (either re-ligated or not) and 
no blue clones were obtained when transforming the ligated PCR products.
Anyway this method is longer to perform and should be only used, I think, 
when your cloning does not work with one of the 2 first methods. With 
methods a and b, the same proportion of white/blue colonies were 
obtained.

Hope this help

Pascal






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