that's correct but the guy i got this system from told me that it's more efficient for the next step. that is from his own experience and he can't have a reasonble reason for that, it works for him. i haven't tried it yet, i guess i'll order my sh-oligos in the next week... i hope it will works for me
that is going to be my next step too, i'm not sure yet about the protocol completly but i think u should add T on your sense strand "CCGGT....." and on your antisense u should add A at the end of your seq. and that is because of the restriction enz u r about to use. if i rememebre corectly it's age1 and ecor1, so be aware of that
I am trying to generate an shRNA vector in the pLKO.1 backbone, and I'm getting stuck at the very first step: oligo annealing.
The capital letters indicate nucleotides that were recommended by the Addgene protocol for designing shRNA oligos for pLKO.1, and the lowercase letters are the sequence targeting my gene. I have five pairs of oligos, each differing in the targeting sequence.
I have tried three different annealing buffers, I tried the waterbath and the PCR cycler approach (95C for 4 min, 70C for 10 min, cool down to room temp over the course of several hours), and I can't get annealing to work.
I am checking the annealing reactions on a 3% agarose gel. No matter if I run the ss oligo, or if I run the (presumably) ds oligo after the annealing reaction, I see the same band pattern. (I should expect to see a slower-migrating band after annealing, right?)
Another weird thing is that the oligos themselves, without any annealing buffer or heating, show up as two bands on a 3% agarose gel. I am attaching a picture of the gel. You can see that for three sets of the oligos (#1-3), they run predominantly as an "upper" band, and for two sets (#4-5) they run as a "lower" band. I also tried combining the sense and antisense oligos (just in ddH2O, no annealing) and running the mixture on the same gel. You can see that the mixture also has a two-band pattern.
I am utterly confused as to what's going on. Any advice would be greatly appreciated.