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Transformation help! Urgent


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2 replies to this topic

#1 netnus

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Posted 21 September 2004 - 05:27 AM

Dear everyone,

I just try to transform the supposed ligated plasmid into XL10-Gold cell strains. But I can't see any colony growing in the LB-Agar plate.
Since the concentration of the ligated plasmid is very low, I can't see any bands in the agarose gel. Some people sugguest me to continue transformation.
The cell I buy is from Strtagene. I use the protocol they give us to transform>
Can anyone tell me what's wrong in the transformation and What I can do in the next step?

Thank you very much!

netnus

#2 jadefalcon

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Posted 21 September 2004 - 07:49 AM

you should do a positive control on your transformation process, i.e. try to transform a whole plasmid into your competent cell as you would do for your ligated product. You can try 10ng, 100ng and 1g of plasmid for transformation, and if your cell are compentent and your transformation works, you should see the whole plate covered with colonies at 100ng plasmid DNA. If there are no colconies in the 1g transformation, something's amiss with your cells and/or your transformation protocol.

since you use commercially available cells, it is rather unlikely that the cells are are a problem, but you knever know.

and maybe if you give us some details about your transformation routine, people can better think of a solution for your problem.

mike

Edited by jadefalcon, 21 September 2004 - 07:52 AM.

--- He who finds typos may keep them! ---

#3 netnus

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Posted 21 September 2004 - 10:56 AM

you should do a positive control on your transformation process, i.e. try to transform a whole plasmid into your competent cell as you would do for your ligated product. You can try 10ng, 100ng and 1g of plasmid for transformation, and if your cell are compentent and your transformation works, you should see the whole plate covered with colonies at 100ng plasmid DNA. If  there are no colconies in the 1g transformation, something's amiss with your cells and/or your transformation protocol.

since you use commercially available cells, it is rather unlikely that the cells are are a problem, but you knever know.

and maybe if you give us some details about your transformation routine, people can better think of a solution for your problem.

mike

Thank you for the reply!
The problem for me is that if the concentration of the ligated plasmid is very low, is there a minimum amount of plasmid to be added to make the cells grow?


netnus




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