Hi All,
I am trying to get some colonies after the bp reaction but I have been trying this for a while and at this point I have no colonies. I am using the pDONR 221, the fragment is 2.3 kb and the concentration after gel purification is 33 ng/ microliter. I am using NEB bacteria. The incubation period has been 18 hrs. I am following all the steps as in the manual but I do now know what I can do to improve it. You think that the concentration after gel purification is ok? If you have some ideas, I'd really appreciate it.
Have you checked if your competent cells are working? Are they efficient? Try just transforming 10ng of mini prep and check if you get a plate full of colonies. You could also try extending the incubation period.
I don't like concentrating the DNA from a gel purification as it also concentrate contaminants that could interfere with the BP reaction. I would suggest to use only water and not concentrating it.
When I had problems with BP cloning, I solved it by using other strains of E.coli: DH5a or TOP10. Top10 worked best for me.
Good luck .
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