Need help about my subcloning pET15b. - (Sep/04/2007 )
Nowdays, I am working with pET15b (5700bp) and my insert DNA(750 bp). I want to subclone my insert DNA into pET15b, but I can not get my construction plasmid for one month. I used NdeI and XhoI to digest pET15b and my insert DNA. The two restrction sites of pET15b are too closed so I firstly used NdeI to digest it and after that I used XhoI to cut it. After the diestion, I used phenol/chromosome to extract the DNA from the digestion solution. I made postive control (pET15b) and negtive control (only vector DNA are in the ligation solution without insert). After transformation, my postive control is very good. But my negtive control and ligation are same that only 3-5 colonies grew on the plates. I checked the colonies but all the colonies are empty.
I did it for one month. So please big one can give me some advices.
I had solve this problem. I did not use PCR product directly buy use TA clone to get the right plasmid.