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anyone working currently on gateway cloning???? - (Sep/30/2009 )

Hi alll

is anyone currently using the Gateway Cloning system? I am having some issues and will be helpful to discuss with someone who is using it these days.



I just started using it. I'm not really buying into how good or easy or useful its supposed to be to move your gene of interest into multiple clones. However, the gene we're working on has typically given us poor expression in E coli and we're getting very good expression with pDEST in BL21 cells.

So far, trying to get some other genes into an entry vector, I'm running into a wall. Trying to transform my pcr into their TA vector (it's pcr8-gw-topo) I'm getting a lot of false positives - colonies that contain the plasmid with no insert. This is after verifying that the pcr is solid. I've also never had trouble with TA kits from other vendors, so no idea what the issue is.

I'm going to try gel purifying the pcr to see if that helps. Otherwise I may end up trying a different entry vector.