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No colonies after LR recombination

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RAD
India

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Status: Frog Egg
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Topic Started by RAD
on 4/20/2011 20:30 PM   
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Hello. iam really new to molecular biology. recently, I performed the LR reaction but got no colonies of E.coli. I tried using a new batch of clonase II, leaving the reaction overnight, it didnt work. any suggestions are highly welcome..thanks in advance!
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Pippuri
United States

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Posted By Pippuri
on 4/21/2011 16:48 PM   
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 Hi RAD,

Have you confirmed whether you are plating the transformed E. coli on the correct antibiotic containing plate? Almost all of the destination constructs that I work with carry Amp resistant gene.

Also, be sure you follow the transformation protocol "precisely" come along with the invitrogen kit- for instance, the incubation/heat shock time, about thawing the competent cells on ice, about not pipetting the competent cells up and down etc.

Good luck.
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