Vector named SST13, map attached, I am trying to clone pri mirna into kpn1, Xba 1 site. i have already cloned dsred into ecor1 site of this vector.
I choose this site as there was Spr of mosquito cloned it already by some other lab, so i thought it would be easy to release and put the fragment of interest.
However, I have been unsuccessful.
I can see the release of spr, when i treat vector with kpn1, xba1.
I tried putting direct pcr products after gel extraction n subjecting to digestion. I could see no colonies on plates.
Then i cloned all pri mirna's by TA cloning as suggested in this forum by others.
Now when i release them n put them into SST13 vector, i still get no colonies. same comp cells seem to work well for others:(
Please suggest, what must be going wrong? ur suggestions will be of great help.