what's the best way to clone PCR products?

Angel Zarain angelz at salk.sbrc.umanitoba.ca
Thu Sep 29 16:46:33 EST 1994


In article <36bjrlINN1ubq at sat.ipp-garching.mpg.de>
krasel at alf.biochem.mpg.de (Cornelius Krasel) writes:

> Mike Preston (preston at warren.med.harvard.edu) wrote:
> :       For those of you who have had success direct cloning of PCR products do
> : you have a step that gets rid of the Taq? 
> 
> Proteinase K digestion, as has been pointed out already several times by
> diverse people.
> 
> --Cornelius.
> 
> --
> /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */
> /* email: krasel at alf.biochem.mpg.de                 fax: +49 89 8578 3795 */
> /* "People are DNA's way of making more DNA." (Edward O. Wilson, 1975)    */

There are several factors that influence subcloning of PCR products.

make sure that the primers used for PCR are 5'-phophorilated,
alternatively, phosphorilate the product using T4 kinase. Also is
important to remove extra nucleotides introduced by Taq pol. using T4
polymerase and the four dNTPs.

For more info see BioTechniques August 1994.

Dr. A zarain-Herzberg
Univ. Manitoba
Canada



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