anybody using GalK cassette for BAC mutation? - (Jul/07/2007 )

I need some discussion. Thanks.

yes, using it at the moment- very unsuccessfully!!

I've only had 3 tries so far (been too busy to focus fully on this project), but no luck. Others in my lab have also had no success with this project. Another group nearby have been using it successfully, but need to screen up to 100 colonies post transformation as the galK system is apparently quite leaky.

How are you finding it??

hi, me too.
I tried it about 8 times now. Non success. Using the same bacteria, SW105, I successed to do mutation using Kan cassette.
While the protocol from NCI said it is very efficient. And I recently read a paper from a Germany lab, who followed the protocol from NCI and said the same thing. Very efficient for them.
Screen up to 100 colonies? If that can give me a single success BAC, I would do it.
But I doubt about it.
Do you see large colony on M63-Gal plates after three days?

I'm using SW102 and I don't see any colonies at day 3, mine start to appear after about day 5ish. What temp are you incubating at??

Hi, me too. I grow them at 32 degree.
Colony begin to appear at 5 days.
How about your MacConkey plates?
I can always see bright red colony, but none of them is the right mutant.

I haven't been doing the MacConkey agar step, I've screening by PCR.