PCR product cloning
Paul W. Diaz, Ph.D.
ghabets at onyx-pharm.com
Fri Apr 25 07:18:54 EST 1997
Try the zero-blunt cloning kit from invitrogen and you will
never have a problem again provided the following:
1. Generate your PCR amplicon with Klentaq (Clontech).
2. Resolve your amplicons on a regular agarose gel (1% TAE
LE agarose gel, LE agarose is available from FMC corp.)
3. Purify your amplicon from the agarose with the qiaquick
spin column protocols for agarose gel extraction (Qiagen).
Use 10 mM Tris, pH 8.3 for the elution NO EDTA
4. Ligate the PCR products as described into the zero-blunt
vector as specified by the manufacturer.
5. It always helps to EtOH ppt the eluted amplicon with
5-20 ug of glycogen followed by resuspension in 10 ul of
ddH2O, use 7 ul in the ligation reaction.
More information about the Methods