Problems in RNAi construction by using pUC19
Posted 02 November 2012 - 01:49 AM
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.
Posted 04 November 2012 - 10:24 PM
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.
Posted 05 November 2012 - 12:58 PM
Is the antisense the same/complement to the sequence as is already in the plasmid?
Details are the key for these sorts of thing...