Problem with Large vector & very small insert - (Dec/15/2013 )

Hello everyone,

I'm having a hard time cloning a small shRNA-coding DNA (size 110 bp) in a really large vector (size 11,68 kb). I have already failed to see colonies using 3:1 and 6:1 insert:vector ratio but I saw in a related, older post that in cases like this, one must use an overdose of insert, for example 2 mg insert for 50 ng vector according to the post, in order to achieve ligation. Does anyone knows if this is accurate?

Also can the temperature of 80 degrees (for heat inactivation of the enzymes) affect the quality of the vector or insert and therefore the procedure that follows? If that is the case would it help if I drop to 65?

I'm using two endonucleases, doing partial digestion of the vector with one of them, so now I'm checking again the controls of each reaction.

If you are failing to see colonies, then I would immediately suspect your transformation and competent cells, rather than your ligation. With competent cells, you always see colonies, just not necessarily the ones you want to see. Are you sure your antibiotic is correct? Try transforming with serial 1:10 dilutions of your original plasmid. You should be able to get at least 10^8 cfu/ug of plasmid. If you can't achieve near this level, then it is your competent cells that are the problem.

Very large amounts of insert is a bad bad idea. You wil (under the best of circumstances) get concatamers of your insert. Worst case, you production of circular plasmid which transforms will go way down.