subcloning

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Shirley
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subcloning

Post by Shirley » Fri Feb 10, 2006 3:47 am

Hi,
I think I have cloned a 1.8kb insert into a 2.8kb vector using two restriction enzymes. When I plated a ligation reaction containing vector + insert on a LB + Amp plate and another ligation reaction containing only vector, I have a colony on each plate. What do you think have gone wrong? By right, I should have more colonies on the plate containing vector + insert.
Thank you.

sdekivit
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Post by sdekivit » Fri Feb 10, 2006 8:43 am

the vector wasn't inserted (restriction enzymes work properly?)

--> to examine whether you have an insert in this case (you're not sure if you have a vector with insert using this method) you can isolate the plasmids en run them on a gel using the same restriction enzymes and see if you find a different bandpattern on gelelectrophoresis.

Good luck with your transfectionexperiments.

rexarunraj
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Post by rexarunraj » Tue Feb 21, 2006 3:52 pm

I am not sure where the amp gene is? vector back bone or the insert?

Amphicillin is not active at 37C after 18 hours.

you may check for the plasmid

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