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Cloning/Transformation question

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Cloning/Transformation question

Postby coppcw08 » Fri Nov 30, 2012 12:24 am

Hey all,

I'm a noob at all this molecular stuff so I'm hoping you all can help a guy out.

I ran a PCR today to amplify some genomic DNA that I will be cloning into a plasmid and transforming into some chemically competent E. coli cells.

I was wondering what I should run as a negative control? I know if the cloning works the plasmid ligates and an antibiotic resistant gene on the plasmid starts to be expressed. If E. coli cells take up the plasmid they will obviously grow on the antibiotic plates I made. What can I run as a negative control to ensure the cells actually took up the plasmid and didn't just spontaneously mutate and ligate and express the antibiotic resistance??

I'm stumped.
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Postby saraelesawe » Fri Nov 30, 2012 8:22 am

I think if u run the transformed E-coli cells in another antibiotic plates as a negative control.
I hope this idea could help you
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Postby JackBean » Fri Nov 30, 2012 10:12 am

you can either transform them with nothing or transform with plasmid with resistance to other antibiotic and then plate on two plates
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby Cat » Sat Dec 01, 2012 12:50 am

The proper way to make negative control is to set up a ligation of your digested vector with water (or TE buffer/elution buffer you are using) in place of insert DNA and use that ligation as negative control in transformation. That gives you an idea of the background (self-ligated vector/contamination) present in your experiment.

What Aelesawe wrote is completely wrong. What Jackbean suggested (transform using water in place of vector) would be valid negative control, but essentially useless and uninformative - unless your antibiotic does not work anymore. The other suggestion - transformation using wrong plasmid (another antibiotic resistance) - is also wrong and has no value.
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Postby bravebeaker » Sat Dec 01, 2012 7:00 am

also sometimes when i ligate short oligos, i use different inster:vector ratios into different plates just to see the pattern.. hope it helps too!
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