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Cloning bacteria: no growth

Cloning bacteria: no growth - Molecular Cloning Forum

Cloning bacteria: no growth - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 08-27-2010, 05:49 AM
Pipette Filler
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Default Cloning bacteria: no growth



Hi

I am cloning competent E coli (tranformed with DNA ligated plasmid) on agar plate with ampicilin (50ug/mg) + X-gal/IPTG. There's no growth (blue/white) on my plate. Even when i use control plasmid with and without insert, there is no growth! What could be the problem?
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Old 10-01-2010, 07:12 AM
Pipette Filler
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Default Re: Cloning bacteria: no growth

1. It could be the competent cells have been thawed. Buy a new batch and test the control plasmid with them.

2. Your LB plates might contain too much antibiotic. Are you sure you added the right amount?

3. Find someone in your lab or building who has cloned successfully, then watch every step of what they do. If they use different volumes of cells to ligation, or a different waterbath, or different tubes (sometimes microfuge tubes can bind DNA), copy what they did and try again.

Good luck!
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