site directed mutagenesis - point mutagenesis of target gene (Aug/08/2005 )

hello everyone

I am a master student of molecular biology. I am planning to Point mutagenesis of nifH gene cloned in a pUC19 derived plasmid called pEN332.

I need help regarding the stratergies to design the primer for this purpose and also literature if you have any.

Hope to hear from you soon

Prajwal
prajbio@gmail.com
Bremen
Germany

---

Hi,
I usually use Stratagene Quick Change PCR-based mutagenesis system to perform site directed mutagenesis. You can go to the web site of Stratagene and download the manual. I think the manual is very clear to let you know the principles of site directed mutagenesis. Then, when you know how to design primers, and PCR conditions, you can only purchase the PfuTurbo DNA polymerase (Stratagene) instead of a whole kit.

---

hi saltycookie
the stratagene manual is really helpful
have a nice day

prajwal

---

I may be biased (the paper is mine ), but the following PCR only site directed PCR approach is simple and very robust.

Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h

Daniel

DNA sequencing protocols

---