Purification of PCR fragments for cloning

ID: 89

Group:

PCR

Prepared by:

Bruce A. Roe (broe@uoknor.edu)

Detail:

After an aliquot of the PCR mixture is analyzed on an agarose gel, the remainder of the reaction is concentrated by ethanol precipitation, resuspended in buffer, and subjected to a simultaneous fill-in/kinase reaction with the Klenow fragment of E. coli DNA polymerase and T4 polynucleotide kinase, the four deoxynucleotides and rATP (34). The reaction then is loaded onto a preparative 1, 1.5, or 2% low-melting temperature agarose gel, depending on the size(s) of the fragment(s) as determined above, and after minimal separation is achieved between the product(s) and the excess primers, the DNA fragments are excised and eluted. After concentration by ethanol precipitation, amplified DNA fragments are ligated into blunt-ended cloning vector, such as SmaI-linearized, dephosphorylated double-stranded M13 replicative form or pUC.

Year prepared: NULL

Date uploaded: 2010-02-26 14:29:17

url:

Laboratory Protocols