boyana Posted January 23, 2007 Share Posted January 23, 2007 Hi! I am currently trying to perform an LR recombination reaction between the positive control of the LR reaction kit pEntr-Gus(donor vector) and the entry vector pDest32. I am trying to perform this reaction successfully and then try to do it with the actual cDNA library, which is already put in a donor vector. For some reason I cannot make my positive control work. I believe that it is a problem with the LR reaction, and not the transformation of the reaction into DH10B cells, because my transformation of the cells with the positive control pUC 19 works perfectly. It is just that they cannot be transformed by the DNA in my LR reaction. Could it be that all the cells are killed during the electroporation process because of high salt content in the LR reaction( I do an ethanol precipitation before I put it into the DH10B cells). What is the most effective way to get rid of the salt? Could it be another problem with the LR reaction, that I just can't put my finger on? I have been trying to do this several months now. Please help. Thanks to everybody. Link to comment Share on other sites More sharing options...
Bluenoise Posted January 28, 2007 Share Posted January 28, 2007 That's easy to answer. What's the Time constant from your electroporation? 4.8 is ideal. However typically anything from 4.4-5.0 will work somewhat. Ethanol percipitation should remove the little salt that's in the gateway reaction, but if you want to remove even more wash your pellet with 70% Ethanol. If I could make a recomendation I'd say to do the following. 1) Make sure that you're doing the LR reaction properly first. 2) Skip the Ethanol percipitation. Just take 1uL of your reaction and use that to transform. The Gateway reaction is so efficient that you'll likely not need to concentrate the DNA at all. (I know they might say to concentrated it to remove things that interfere with transformation, but I usually find this unecessary) Link to comment Share on other sites More sharing options...
AngelReign Posted April 20, 2009 Share Posted April 20, 2009 (edited) The Entry/Gateway® system is a timesaving technique for quickly generating .... To test the efficiency of pGWS entry clones in the LR Clonase II™ .... We successfully corrected this over-expression problem by using the Ubi-63E promoter Edited April 20, 2009 by Cap'n Refsmmat Link to comment Share on other sites More sharing options...
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