Hi, I am trying to clone 2 important proteins in one plasmid. These two proteins are linked together in the host genome. Thus, I tried to amplify these two protein DNA which is about 1kb long. Then, I ligated it with a plasmid and transformed it into a competent cell. However, I didn't get any colony. May I know whether there is any method that can clone this 1kb DNA? I am thinking of ligating this PCR product with a primer that contains ampicillin and Ori or a few RE sites to resemble a plasmid so that I can control the plasmid size. Do you think it will work?

Thanks.

I think first you need to tell us what plasmid you tried to clone your fragment into the first time, which restriction sites you tried to use and if you dephosphorylated your vector after you opened it up as part of of the cloning procedure.

Did you perform a positive control transformation of your empty backbone vector to check it grows in your ecoli and develops resistance to the antibiotic you used for selection?

Your DNA ligase may be off in which case your ligation will never have worked and you would not have got colonies.

Putting a 1kb fragment in to a vector should be a pretty trivial exercise these days.

Outline your cloning strategy in detail and someone here can help you troubleshoot.

Yes 1kb is not big. I am using 1.1kb with flagPCMV2 vector, and no problm. tel us more and maybe we could help.

Thanks a lot. I really appreciate it. This is actually my friend's problem. I will ask him more and get back to you all later. Thanks. Thanks.

Ask your friend to register, it is always best for him to ask by himself.
Guy

Hi, I am back. I am sorry that I have given the wrong information. I would introduce this forum to my friend but it is up to him whether he wants to join or not. Actually, what he is trying to do is to insert 2.5kb DNA (2 proteins) into a vector. He tried GFP Fusion TOPO TA Expression kits, pGEM-T and pBudCE4.1 with the presence of one positive control for each vector. However, he just couldn't get the correct colony. What he is doing now is to clone the 2 proteins separately in 2 plasmids and try to co-transfect them into cells. It works. But, I would like to find out why he couldn't get the clones. Is the DNA insert too large for the plasmid?

Thanks for helping. :)