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Transformation(?) issues


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#1 Shadowstep33

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Posted 22 June 2010 - 09:11 AM

Hey I'm having issues with transformation and cloning.

I've been trying to ligate ampicillin resistance into a vector and I'm not sure what I'm doing wrong. I've digested both, dephosphorylated the vector, PCR'd the plasmid with the desired casette, digest both (though accidentally together but then I ran them on a gel and that seemed to separate them). I've ran controls that lead me to believe it's a transformation issue (I made dephos. Ligase + and ligase - plates with no results on either, I made digested vector ligase + and ligase - plates with no results, and I also did a test on my transformation efficiency because I was incubating in a heat block as opposed to a shaker but my TE is still low even in the shaker with the parent vector). I CAN transform, but apparently with low TE.

During transformation I incubate on ice for 30 minutes, heat shock at 42C for 2 minutes, ice for 2 minutes, recover fat 37C for 60 minutes.

I'm pretty lost and any suggestions/help would be appreciated.

#2 pito

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Posted 22 June 2010 - 09:30 AM

Its always hard to tell what the problem might be, but one hint is to do an extra ethanolprecipitation step after you gel extracted your vector and gene.


And what do you mean with digest both?

you did an digest on the gene too? If there is a restriction site in the gene then your gene is ruined.
(but you say the gels of both are ok?)

You might also wanne try an electrotransformation.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Shadowstep33

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Posted 22 June 2010 - 09:54 AM

im considering trying an electrotransformation next, and yes I digested both the vector and insert together. My supervisor told me both needed to be digested (as far as I know there wasn't a restriction site in the amp-resistance casette).

I think I also may have not been as gentle as I needed to be with the cells, but even with that I was able to clone the parent vector. I'm going to try a shorter heat shock duration on transformation of the parent vector to test if that increases my TE. I'd like to get this finally done though so that I can move on.

I'll also do the extra ethanol precipitation step like you said though I did centrifuge the minicolumn assembly in order to evaporate the ethanol

#4 pito

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Posted 24 June 2010 - 09:40 AM

you can centrifuge all you want: an extra ethanolprecipitationstep is sometimes really needed.

Anyway: try to digest them seperately (so you can put them on different gells or different lanes at least). And I would also try an electrotransformation.

And you are sure you used the correct restriction enzymes?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





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