Competent cell efficiency

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Competent cell efficiency

Postby xtnw58 » Sep 27 2016 4:43 am

Hi,
I've produced Top10 competent cells with the CaCl2 method but when I then used them for a transformation I've obtained only 1-2 colonies (!!).. this was after 16 h of incibation at 37C, should I wait longer?
Because during the transformation, when I spin down the cell culture after the 1 h incubation step at 37C , 200 rpm, I've observed an extremely tiny cell pellet ---> no much cells grew.. If I plated such a small amount of cells, should I wait longer for the cell colony to show on the plate or if I'll ever get colonies will be contamination?

Of course I have also to remake competent cells, do yiu suggest a protocol in particular for the calcium method?

Thanks,
Xile
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Re: Competent cell efficiency

Postby r.rosati » Sep 27 2016 7:34 am

Did you transform with a stock plasmid specifically to quantify transformation efficiency? Or is this transformation part of a research project?
The cell pellet might very well be small; this is not a problem per se.
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Re: Competent cell efficiency

Postby xtnw58 » Sep 27 2016 10:03 am

This is prt of a research project. I' m making stocks of plasmids I inherited from a previous student.
If I've got not particularly (apparently!) competent cells that I' m using to do the transformation, even if I get a few colonies can I still use them or you think it's contamination?
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Re: Competent cell efficiency

Postby r.rosati » Sep 27 2016 10:19 am

I understand your concern; I think you could test the colonies to see if they match with the plasmids you're expecting them to be.
First, you already know that they grow on the correct antibiotic (you could also plate competent cells with no plasmid to see if you get any contaminant colony).
Then, every plasmid should have a certain size, produce a certain pattern of bands on a restriction digest, amplify with certain PCR primers a band of a certain size, and so on. See if you can settle on a test for each plasmid, that will make you feel confident you have the right colony. I mean, if you knwo that there are two plasmids with an insert that differs by one nucleotide, and you're worried they're mixed, you might as well sequence them.
I could share a horror story of two of "heirloom" plasmids that were not what a couple students thought they were, and the time they lost with them. So better settle it out.
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Re: Competent cell efficiency

Postby r.rosati » Sep 27 2016 10:21 am

Oh and about the low amount of colonies in general... it might be due to the cells. maybe you could transform with a known amount of pUC18 or pUC19 and calculate their efficiency.
Also, if you are transforming with plasmid DNA eluted from Whatman paper, in my experience you do get fewer colonies than expected.
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Re: Competent cell efficiency

Postby NikolayGoncharov » Sep 08 2017 12:19 am

Hi,
Does any one know what is the best way to transform native Bacillus pumilus strain? How to make cells of this strain competent to electroporation?
Thank you.
Nikolay
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