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Screening colonies after transformation


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#1 spellberg56

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Posted 10 March 2010 - 09:31 AM

Hello, I transformed some chemically competent cells the other day with intact plasmid. In other words some one already did the ligation and screening of positive clones, prepared pure plasmid DNA and sequenced the plasmid. I don't know what the amount of plasmid I used for the transformation was but probably 100s of ng. I grew the cells on LB+ampicillian. Is there any reason why I should have to screen these colonies to make sure they have the plasmid in them? I'm using these cells for a protein induction and I just took one colony randomly off the plate to grow up.

Thanks

#2 Denny

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Posted 10 March 2010 - 10:17 AM

Hello, I transformed some chemically competent cells the other day with intact plasmid. In other words some one already did the ligation and screening of positive clones, prepared pure plasmid DNA and sequenced the plasmid. I don't know what the amount of plasmid I used for the transformation was but probably 100s of ng. I grew the cells on LB+ampicillian. Is there any reason why I should have to screen these colonies to make sure they have the plasmid in them? I'm using these cells for a protein induction and I just took one colony randomly off the plate to grow up.

Thanks


If the competent cells do not have ampicillian resistance, they will not grow without a plasmid that gives amp resistance. So make sure that your cells are not resistant to amp, then you will know that colonies growing on your amp plate will have your plasmid incorporated.

#3 wizzkid

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Posted 12 March 2010 - 07:49 AM

you should screen them because everyones human and therefore error can occur. I have seen it happen in the lab where someone has gven someone else what the samples/clones/reagents that they believe is "correct". its easily done. Golden rule in science, always make sure YOURSELF that what you are using is correct to remove therefore you can remove it from the equation if an error occurs later down the road with your experiment. (and it will save you alot of frustation) :huh:




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