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Strange results of cloning


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#1 ram

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Posted 17 March 2011 - 01:04 AM

We are trying to clone an open reading frame into pEXP5 CT TOPO vector for recombinant expression in E. coli. In each of the last 4 attempts of the cloning and transformation in Top10 strain, we got very few (2-5) colonies. Strangely, we have observed that all of the 6 recombinant plasmids that we have got till now have reverse oriented insert! By probability, 3 of these should have got properly oriented insert and 3 reverse. Is there any known biological reason (eg. insert-sequence based bias) for the tendency towards cloning in one particular direction?
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#2 HomeBrew

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Posted 17 March 2011 - 02:40 AM

Perhaps the gene is toxic to E. coli in the other orientation (which I assume is the orientation under which the gene would be expressed)?

BTW, your logic is faulty -- the number of colonies arising on the plate doesn't necessarily indicate the number of successful independent ligation and transformation events. There might have been only one successful ligation/transformation event, and the 2-5 colonies you're seeing on the plate are all siblings of one another.

Are your cells sufficiently competent? Whenever you see such a small number of colonies after transformation, cell competency needs to be questioned...

#3 ram

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Posted 17 March 2011 - 03:35 AM

Perhaps the gene is toxic to E. coli in the other orientation (which I assume is the orientation under which the gene would be expressed)?

BTW, your logic is faulty -- the number of colonies arising on the plate doesn't necessarily indicate the number of successful independent ligation and transformation events. There might have been only one successful ligation/transformation event, and the 2-5 colonies you're seeing on the plate are all siblings of one another.

Are your cells sufficiently competent? Whenever you see such a small number of colonies after transformation, cell competency needs to be questioned...


Thanks for reply Homebrew
Yah...I had thought of the possibility of the gene being toxic to E coli. But in this particular vector, the gene is under the control of T7 promoter and the Top10 strain doesn't encode for the T7 polymerase; whereas other strains such as BL21(DE3) have T7 polymerase and I assume that only under such circumstances the gene of interest will be expressed. Do you mean toxicity by any other (non-proteinaceous) means?
You have made a very good point that each colony may not represent an independent transformation event. In our case we have got 6 recombinants in 4 independent ligation and transformation experiments. So can't we now expect that at least 1 of these 4 experiments, if not more, should yield the plasmid carrying properly oriented insert?
Yah..we too are doubting competency of the cell since a long time...we'll check it.
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.




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