TA cloning help - (Nov/04/2006 )

Hi I came across a problem with my TA cloning. I PCR amplified my insert which is about 1kb using primers I desighed. The cDNA amplification was fine. Then I cut out the bands and genecleaned the products followed by ligation into TA vector. But after transformation and overnight growth, there were many satellite colonies coming up on both the ligation plate and the control plate and there was no apparent difference of the number of the colony between the ligation plate and the control plate. Could anyone give me some suggestion? That would be very helpful for me. Thanks a lot.


Nan

The satellite colonies are due to the ampicillian going bad in the plates. Make new plates use amp conc about 100-150 ug/ml. You can also use the blue white selection with the TopoTA vector by covering the plate with IPTG and X-Gal according to the instructions. You will pick the white ones since that contains an insert.

TA cloning relies on the fact that Taq amplified DNA has a T overhang and an A overhang. By design, the vector also has a T overhang and an A overhang, to allow it to capture the insert.

These overhangs on the vector also allow it to self ligate, so you need to use more than ampicillin selection -- you must differentiate between transformant colonies containing vector only and those that contain a successful clone. Usually this is done with blue-white selection on Xgal plates, as tap14 suggests.

BTW, I've never had an ampicillin plate "go bad". It's likely you were not seeing "satellite colonies" (colonies that break through the ampicillin selection because of a zone of degraded ampicillin around a true colony due to the action of beta-lactamase), especially after only overnight growth.

Amp plates go bad when an undergrad adds the amp before autoclaving the media.

Thanks for all your recommedations. I then when on following your suggesion to use Blue-Write selection. There were some white colonies appearing on my recombinant plates comparing with the control (only TA with water and no white colonies on it), but after the colony PCR, I still could not find any positive clone on the gel while control PCR worked ok. I repeated my experiment twice and turned into relatively same results. Could you give me some ideas

Thanks