transformation after miniprep - (Oct/29/2008 )

Hello,

I have a pure solution of 1 specific plasmid (after miniprep) and I want to make a glycerolstock. The problem I have is that transforming doesn't want to work :-S.
I transformed it and no colonies appeared :-s... isn't that strange?
Does anyone have a suggestion what can be the problem here?
Thanks

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what about your competent cells?and concentartion of antibiotic? are they correct ?

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Yes, I've done the procedure with other plasmids, and that worked. So, one doesn't want to work and I don't know why

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You have used the same lot of competent cells and got colonies with other plasmids? I ask because invitrogen just sent out an entire lot of Top10 cells that were bad. Had one lab member burn through an entire box thinking the ligations weren't working. I tried to amplify a pure plasmid that sequenced just fine and got no colonies. Retransformed same DNA prep into NovaBlue cells and got hundreds of colonies. Invitrogen is replacing the entire order.

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What has happened to the original transformation plate? The one you used to grow up your miniprep? Just grow up some more of them.

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The original plate contained just two colonies. I picked both of them to see if my gene was correct incorporated. Now I know that it is correct, I just want to make a glycerolstock of them... but retransforming doesn't want to work.
And yes I've used the same competent cells, and other plasmids could be easily transformed

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how about cleaning up the miniprep DNA with phenol-chloroform and then re-transform.

Check that you do indeed have DNA in the prep, the competent cells work, and your are using the right selection plates.

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I've tried it again after measuring the concentration again. And now I've had 5 colonies, not much but enough to make a glycerolstock
But strange that it doesn't give hundreds of colonies he?

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