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Cloning a foreign gene into E-coli

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#1 Enzy



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Posted 22 January 2011 - 10:15 AM

Dear Friends,
I am facing the problem with cloning. I have insert with 2.6Kb to be cloned into 13Kb E-coli vector . Vector has cloned strong promoter which is derived from the source same as the insert coming from. According to the strategy the desired will be cloned in front of promoter using PCR product digested either with same enzyme at both the ends(NcoI) or two enzymes(NcoI and SmaI). After digestion,ligation and transformation I got following results,
1) when digested with both the enzymes i did not get any colonies after transformation.
2) When NcoI is used I got clones but the orientation is opposite to the promoter(this is not a ideal case).
Then I removed promoter and cloned the gene with upstream region(looks like there is promoter like region)
then I got the clones without insert. So i have following questions(u can say doubts),
Is this gene is toxic to E-coli?
when I used the clones in which the gene is in opposite direction and the mutant is complimenting phenotype. So can I use this construct in future as the complimented strain( ideally its not possible becoz gene is sitting opposite to the strong promoter)???
Is ter is any other possibilities to clone the gene in proper orientation?
Please help me with this problem
Thanks in advance
with regards

#2 perneseblue


    Unlimited ligation works!

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Posted 22 January 2011 - 08:20 PM

Assuming that you are using a prokaryote promoter, overexpression of your gene maybe toxic

But just to be complete , there are two possibilities.
1 - ligation not working, probably due to incomplete digest by SmaI
2- gene toxicity.

Still... did you do a serial digestion when cutting with NcoI and SmaI? SmaI enzyme has to be incubated at 25 Celsius. Its half life at 37C is only 15min.

In future, I would recommend XmaI as an alternative to SmaI. Both enzymes recognise the same restriction site. XmaI however can cut at 37 C and produces a sticky overhang rather than a blunt end (which SmaI is produces).

If toxic gene expression is a problem, you should use an inducible promoter. This should solve the problem
May your PCR products be long, your protocols short and your boss on holiday

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