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Unable to clone in E. coli cells

DH5a transformation SF9 insect cells

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3 replies to this topic

#1 Doda

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Posted 01 November 2012 - 12:38 AM

Hi, everyone!
Does anybody know why some transcripts cannot be cloned in E.coli cells?
I have tried cloning the cDNA of a transcribed gene using DH5a competent cells, and whenever I would transform them I wouldn't get any colony. I later checked for further information about that gene, and found out that the very few groups that could clone it, they did in SF9 insect cells.
I succeeded, however, in cloning either the N- or C-terminus of that same transcript.
Any idea?

About my materials: pcDNA3.1 vector + ~4.1Kb insert.

Thank you.

#2 HOYAJM

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Posted 01 November 2012 - 07:05 AM

It sounds like the gene product is toxic to E.coli. Even if you have not induced expression of this gene, there could be leaky expression that is sufficient to kill the cells. From the information you have obtained, the N and C termini are not lethal by themselves. This is not uncommon and there should be plenty of literature on methods to express toxic genes. Take a look at this paper:

http://www.ncbi.nlm....pubmed/16472168

Edited by HOYAJM, 01 November 2012 - 07:07 AM.


#3 John Forsberg

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Posted 01 November 2012 - 06:26 PM

For expression of toxic proteins, a good method to try is Studier's autoinduction media. The cells are grown overnight with glycerol and glucose present to suppress leaky expression, then diluted into the autoinduction media (has glycerol, glucose and lactose for induction) to auto-induce expression only when the cells are at saturated growth density.

The cells basically kill themselves at the end, but you'll get some protein out of it (usually).

http://www.ncbi.nlm....pubmed/15915565

Very handy system for toxic or non-toxic proteins.

#4 ascacioc

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Posted 04 November 2012 - 01:43 PM

Even if you express a protein in Sf9 insect cells, you still need to clone it in a vector using bacteria. Are you using SOC media during recovery? You have a large insert; this is more difficult to clone; are you using commercial competent cells or do you have them self-made? How old are they?

Andreea





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