how can i find the positive clone analysis of TOL2 transposon plasmid constructs, i could not find the right clones even after sequencing using the appropriate M13F and M13R primers, even also by RE digestion, anybody could you please suggest ??
Please specify the problem, do you want to modify the insert for transgenesis an do not get the right clone?
Yes sir, I want to develop a transgenic animal, for that I m using my gene specific promoter in 5' entry vector. Tthe EGFP and Poly A as Middle and 3' entries respectively. These entry clones are recombined into a Destination vector. Now I could not get the positive clones even after multiple trails using different confirmation methods. suggest me please.
Dear Chakrapani,
I suppose you are using a 3-fragment gateway cloning strategy. Unfortunately I am not familiar with this vector system. I would propose to evaluate the clones by colony-PCR screening using primers matching the different cassettes, that could speed up the identification of complete or partial destination vectors.
Alex
Sir,
I'm following Dr. Km kwans tol2 kit and yes as you said it is a multiple gateway cloning system. I have tried colony pcr too. I m using LR clonase recombination enzyme which is effectively works. Okay sir let you know the results, this time again I m repeating the entire reactions. Thank you very much sir.
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