Have you performed the single digests to make sure your vector is being cleaved by both enzymes? Are you performing a double or sequential digest? Sometimes it can be difficult to get efficient cleavage when the RE sites are close together, and be advised that BstEII requires 60C incubation temperature, not the usual 37C.

Are the oligos encoding the shRNA designed to produce overhangs or do you digest these oligos prior to ligation?

I am pretty sure that my double digestion is working pretty well. I ran the digestion result on gel and it showed 2 clear bands with correct size. The picture is attached.

I designed an overhang shRNA oligos corresponding to BglII and BstEII. I thought I just need a simple ligation step to do it but after several tries I still can't get it.

Now I'm thinking of designing another shRNA fragment with an A overhang ends which is compatible with TA cloning vector such as pGEM T Easy. However, I need to explain my problem to my boss before ordering another shRNA.

I don't know whether the shRNA is properly annealed as the I ordered it in annealed form. Could it be a possible reason for my problem? Do you think it is possible to check the oligos on agarose gel?

Are lanes 1 and 4 some sort of MW marker? Can you provide more info about what is in each lane of your gel?

Lane 1 and 4 are lamda hindIII ladder.

Lane 1 is uncut pCAMBIA1305.1 plasmid

Lane 3 is pCAMBIA 1305.1 plasmid cut with BglII and BstEII giving 2 bands, 9796 and 2050 bp.

I purified the upper band (9796 bp) and then ligated it with my shRNA, but until now still get nothing.

Please give some advise

thanks

When you say you get nothing, do you mean you get no colonies after transformation with your ligation reaction, or you get colonies, but none are recombinant? How many colonies did you screen and by what method? If your ligation is inefficient, you may have to screen dozens of colonies. In addition, it may be worth trying to reanneal your shRNA oligos in a thermocycler if you don't trust that your commercial vendor did this step properly.

How much of your oligos are you adding to the ligation reaction?

I did not get anything growing except for E. coli transformed with pCAMBIA 1305.1. That is why I think the problem is with the ligation.

Do you think it is possible to reanneal my shRNA after dilution? I've diluted it to 100 micro molar with nuclase free water.

Can you suggest me any recommended protocol for annealing?

I made the ligation reaction based on online ligation calculator.

Vector length 9796 bp, shRNA 121 bp. Vector amount 90 ng (nanodrop calculation 30 ng/micro liter)

I mixed 90 ng vector with 3.3 ng insert.

Can you lead me from here?

Thanks

I usually follow the protocol provided by Addgene, http://www.addgene.org/plasmid_protocols/annealed_oligo_cloning/

You might try a higher ratio of insert to vector (use up to 10ng).