The desired gene to be expressed is aioBA of ~3.1kb . I have already tried pET100/D Topo vector. The insert was PCR amplified (forward primer with CACC overhang at 5’-end) with Pfu Polymerase (to get blunt end product) , gel purified to get a single, discrete band of desired size. After cloning (at recommended molar ratio) and transformation few colonies(5-10) were on LB/Amp plate. Colonies were analyzed by Colony PCR with orientation specific primers (T7 reverse-binds to vector and our designed primer aioBA_exp_F-binds to insert). Unfortunately, no band of desired size amplicon were there. I also miniprepped some transformant colonies and restriction analysis was done using PstI (cut once in vector and once in insert) but digestion pattern suggesting an empty vector. So far, I am assuming that the ligation of vector and insert is not happening. I have done cloning for several times, but was never successful.
So before I switch to another vector, I want to get some suggestions.
Thank you in advance.
You can increase the molar ratio of vector:insert of 1:3 up to 1:5 to increase the chances of ligation or you can try the Hot-Fusion method Article Hot Fusion: An Efficient Method to Clone Multiple DNA Fragme...
I think the fact that you have so few colonies is the problem. Either one of the components in the ligation reaction is problematic or your competent cells are not of the necessary quality to get sufficient transformants. Try some controls to distinguish.
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