Problems in RNAi construction by using pUC19 - (Nov/02/2012 )

Dear All,

I am a new member of this forum. I have a problem in my research. I am trying to contruct RNAi by using pUC19 as a base vector. The first step is to insert part of the gene (500 bp) into the plasmid and use the recombinant plasmid for the next step. The problem rise when I try to insert the antisense gene (700 bp, 500 bp is the antisense, 200 for loop) into the recombinant plasmid (from the first step). The cloning always failed and I already retry over and over again. The result is still negative. I don’t know what is wrong with the protocol. I use NEB restriction enzymes and T4 ligase.
I can always insert DNA fragment (in various occasion) into the pUC19 but always failed in the next step. Please help me with this issue.

Thank you,
Chris D

So what is your protocol then???

The protocol is standard protocol.
The plasmid and insert were digested overnight at 37oC, deactivated at 80oC for 20 minutes, and then the plasmid was treated with CIP for 37oC for one our. After that, I purified the DNA fragment by using gel extraction kit. The ligation is one hour at room temperature and after that I clone it to E. coli.
The problem is when I clone the first fragment, the result is good, but when I tried to insert the second one (to the recombinant plasmid), it always fail. Please help me with this matter.

Thank you,
Chris D

What cloning sites are you using (for both bits,), blunt, cohesive? What strain of bugs are you transforming? Which backbone plasmid is it? Are you quantifying your DNA? What molar ratios and other conditions (4 deg C? 37 deg C?) have you tried for the ligations? How did you confirm the insertion of the first bit? Have you tried fresh ligation buffer?

Is the antisense the same/complement to the sequence as is already in the plasmid?

Details are the key for these sorts of thing...