E. coli transformation - (Sep/21/2005 )

Hi all,

I am using the Quickchange site directed mutagenesis kit for mutating a ~ 4.1 kb plasmid. After this I treat the mutated plasmid with DpnI to digest template methylated plasmid. But I have real problems transforming E. coli with DpnI trated plasmid. Why?

Any suggestion will be very appreciated

Thanks in advance

---

What exactly is your problem? Are you seeing no colonies at all? And if so, are you doing a positive control with the same vector (just not DpnI treated)? Are you sure your PCR is working? Are your cells competent?

---

Thanks Vairus,

Amplification seems to be well done as seen on agarose. Cells are competent. I see no colonies after transformation procedure. Control with template non mutated non DpnI treated plasmid allows good number and quality colonies formation.

---

Have had similar problems myself. But no problems since I stepped up to using rubidium chloride competent cells. Thats why you get supercompetent cells I believe. Some vectors are required in high amounts or as I found with the Duet vector, the cells did not like them that much. You need to purchase more competent cells

---