I have been trying to clone an shRNA for a gene knock-down in pLKO.1 vector but I am not able to get the clone. I don't know what the problem is. I have tried annealing the oligos at 95 C in a PCR machine and then cooling them gradually to room temperature. I also tried annealing in boiling water and progressively cooling to room temperature. But when I am running the oligos annealed along with sense and anti-sense strand, I am getting the same band in all (not upward shift in an annealed oligo). So I think oligos are not annealing in my case. My oligos are desalted and not HPLC purified. Should the oligos for shRNA cloning should be HPLC purified? I am using this addgene protocol for shRNA cloning: https://www.addgene.org/protocols/plko/
What is the formulation of your annealing buffer? If you're not confident with your homemade buffer, you can use 10xNEBuffer2 (or 10xM buffer of restriction enzymes) for annealing buffer.
And have you tried ligation of annealed oligos and vectors?
It is hard to detect the shifting between annealed oligo and sense/antisense oligos by running. You should move on to the next step rather than stop here. Desalting is enough for the purity of the oligos required for ligation with the pLKO.1 vector.
@ Yohei Kono
I used NEBuffer2 only for annealing. I also proceeded with ligation and transformation but I didn't get any colony in that whereas I got colonies in the negative control (where there were no shRNA oligos) showing vector self-ligation. But this self-ligation is not possible as I gel-extracted the 5 kb band of pLKO.1 vector having two different RE site end (EcoR1 and Age1). That is why I am thinking that annealing might be the problem.
OK Goyani, I see your situation.
I don't know if I can help you, since the difficulty of annealing oligos depends on their shRNA sequence, but I have a few suggestions.
1. After heating at 70°C for 10 min, do not cool at room temperature, but use a thermal cycler. Attached is a photo of the conditions I am using, annealing from 70°C to 37°C, at 1°C/2min40sec, over a period of time.
2. When cutting the pLKO.1 vector with AgeI and EcoRI, if you are double-digesting, digest one by one starting with AgeI. In my case, this reduced the number of colonies that were not aimed, so double-digesting may not work due to 3D interference of these enzymes.
Yohei Kono ...Thank you very much for your time and response. I will definitely try what you suggested for annealing and cloning and let you know. I also wanted to ask which E. coli bacterial strain you used, DH5alpha or StBl3?
I prefer to use NEB Stable (a variant of Stbl3?), but, they are difficult to grow with pLKO.1 and we are considering switching to Sure2. And I use carbenicillin than ampicillin for liquid culture (after Amp+ LB plates).
Anyway, I think that something unexpected is going to happen with DH5alpha.
Good luck!
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