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Please save me ! - pAdeasy adenovirus vector system (Jun/01/2007 )

Hi, Guys, is there anybody using AdEasy adenovirus vector system? I have a gene 9.4 kb shuttle vector and try to integrate to pAdEasy adenovirus vector, which is about 30 kb. First I digested with Pme I (originally I suspected the Pme I is old and I ordered a new one), and SAP it ( and I did not inactivate it by 65C). I tried CIP as well. And I did gel purification ( I did see one band compared to uncut, but I was not sure it is linealized DNA). And I did electroporation. Everytime I got colonies from 30 to 200 (varied), and miniprep it. I should see 4.5 kb and 30 kb bands after vector DNA recombinated into virus genome). However, I always get damned 6 and 3 kb bands, which is cut from plasmid vector but not from integrated AdEasy virus. They are never recombinated, never. I am struggling for 1 month. Please help me out!!!!!!. thanks. wacko.gif


Did you use a new AdEasy Kit?? If not:
Be VERY sure it is the shuttle vector. Digest with RE. May be you lost one of the “arms” so you don’t have the homologous sequence for recombination (it happened to a labmate ones, when he got the plasmid as a gift).
Sure it is linear vector (run in a 0.4 or 0.5 % agarosa gel just next to your uncut vector)
Be sure you use kanamicin.
Be sure you have a good pAdEAsy prep and be sure the vector has not modifications.
Be VERY sure you are using BJ (recombination proficient strain).
Mix very well the pAdEasy and the shuttle (I heat at 50-55°C for 5 min and cool at room temp before adding to the competent cells).
Be sure you don’t have contamination:
Control(-) 1: linear shuttle without pAdEAsy (you should have few colonies).
Control(-) 2: pAdEasy (you should have 0 colonies).
Control(+) 1: shuttle uncut.
Control(+) 2: pAdEAsy (in amp+ plates)

Be sure you don’t have problem with the minipreps of large vectors.
When you pick colonies and miniprep, take one or two colonies from the C(+) 2 to be sure your preparations are ok (ones, I had problems with the large vectors preparations, 3-10 kb plasmid preps were ok, but 30 kb plasmid preps don’t).

I hope it could be helpful!
Good luck

-aztecan princess-