Hello everyone,
I has been worked with a Y2H Screening and my cDNA library (custom library from Arabispsis plants - ProQuest Two-Hybrid Sytem Invitrogen Service) was built in pEXP-AD502 vector. So, I receive this library from a colaborator in one tube with Min-prep (~2.000 ng/uL). At this moment, I need to transform E. coli cells and after that to amplify my library and perform my Glycerol stocks. Moreover, I have to obtain a high mass of plasmids for further manipulations such as Yeast transformation and Y2H protocols as well.
CURRENT STATUS: I already have the E. coli TOP10 eletrocompetent cells (10^9 ufc/ug.DNA), however I don't no how many nanograms of the libary I must to use in the transformation protocol and how to proceed with regard to the volume and the number of plates to growth my cDNA clones.
I'm very thankful if anybody could send me some suggestions, tips or protocols to do that.
Best Regards,
Helder Anderson
By a quick search it is obvious that the system you are using (or your collaborators) is a gateway cloning technology and the vector pEXP-AD502 is a gateway expression vector. Thus, you need to follow the protocol and right competent cells. You can not simply use Top10 cells for the transformation. Please buy a suitable kit/competent cells for the transformation and follow the instructions for the screening of the candidate interactor clony for the induced reporter gene. It would be better to consult your collaborators before doing any experiments.
Transformations/electroporations have a limit to how much DNA you should add before you approach saturation. This is usually about 10ng range (although it does vary). So once you are at the saturation point, rather than adding more DNA, it is best to do multiple electroporations to get a larger number of colonies.