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Why did some Colonies Grow?

Why did some Colonies Grow? - Molecular Cloning Forum

Why did some Colonies Grow? - 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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  #1  
Old 05-19-2008, 04:37 PM
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Angry Why did some Colonies Grow?



Why did some Colonies Grow?

i cant understand it! after ligation and plating i only get 2 colonies

any ideas?
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Old 05-19-2008, 06:19 PM
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Default Re: Why did some Colonies Grow?

First thing I would check is the competency of my competent cells (do you have a positive transformation control)?

Without any details on your protocol there are so many steps that could have gone wrong I can't suggest anywhere else specifically to look. Can you give some more details (restriction enzymes, blunt vs. sticky-end, phosphatase treatment? DNA concentrations?)
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Old 07-01-2008, 09:46 PM
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Default Re: Why did some Colonies Grow?

Besides, if any of those colonies contains the correct construct - what else do you want in life? :-)
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Old 07-07-2008, 06:42 PM
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Default Re: Why did some Colonies Grow?

My colonies always grow even though they've never had the right construct. That is strange!!!
sorry, i have no idea. I also have problems with the cloning voodoo thing
good luck
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Old 09-27-2009, 08:05 PM
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Default Re: Why did some Colonies Grow?

Trypsin - are you treating your vector with a phosphatase?
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Old 10-26-2009, 07:25 PM
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Default Re: Why did some Colonies Grow?

trypsin.. if you could give a little more details , that would be helpful.. how do u digest your plasmid and insert ? one or two enzymes?
do you check the digested fragments on agarose gel? to see if the restriction enzymes work well.
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