DH5alpha transformation: protocol - (May/24/2006 )

Hi,

I am trying to transform DH5alpha with a closed vector and have had little success. Other people in my lab use the same competent cells so I know that they are alright.

I have noticed online that peoples transformation protocols sometimes vary in the temp they use for heat shock(i think 38 to 42 from memory) , and the length of time that the cells are heat shocked for (40sec - 90sec).

Does this have an effect on success? I have been doing at 42degrees for 90 seconds, then on ice for >2 mins (up to half an hour), then incubate at 37degreesC for >1.5hrs, then plate out on 20mL LB agar with 20microL of ampicillin (100mg/ml), 100microL of X-gal and 20microL of 450mM IPTG .

Can you think of anything I am doing that might be stopping it from working?

Any assitance will be greatly appreciated

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Hi G-star,

Did you add SOC or LB after the heat-shock?
30min on ice after the heat-shock, I think the transformed cells will not like this and anyway it doesn't have to be that long. 1 min is already enough. I even did it without going back to ice after the heatshock and that was OK too.

This is how I do it:
Thaw your competent cells on ice for 10 min.
Add your DNA to the cells. Don't add too much, for transforming plasmid DNA 10 picograms is enough! In case of a ligation transform half of your ligation or less. Always keep in mind that for transformation less is more!
Leave your cells + DNA on ice for another 10 min.
Put the eppendorf from the ice straight to 37C and leave for 5 min at 37C.
Take out from the 37C and place on ice for 1 min.
Add 500 microliter SOC or LB (no AB!) to your transformed cells, transfer everything to a falcon tube and put on a shaker at 37C for 45min to 1 hour.
Plate out 100 microliter on a LB-plate + AB (+ X-gal/IPTG)

Anouk

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I think anouk is right in that the cells might not tolerate 30min on ice after heat shock.

Also shake the cells after adding SOC or SOB media, not just incubate.


This is my lab's protocol.

I add DNA to the cells, and incubate on ice for 20-30min.
Heat shock at 42C for 40-45sec
Incubate on ice for 1-2min, then add 700ul SOB or SOC,
Shake them 1hr 37C
Plate the entire 700ul on to a plate with antibiotics.

Good luck !!!

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I thought the 30 min might have caused the problem - someone was using the biohazard hood when i went to use it so I was forced to wait or risk contamination...

We use LB media in our lab with the DH5alpha cells.

So you think that incubating with shaking for 1 hour is enough? And heat shock for 40 seconds?

Also I didn't know that you could add too much vector: I added 100microlitres to 100microlitres of cells, another reason why it wouldn't have worked.

Thanks so much for your help everyone, I think this site is really great!

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If you are transforming E. coli with an antibiotic, you absolutely do not need a biohazard hood. If you have autoclaved plates and media, and use an antibiotic, you won't have a problem unless your lab is a dirty basement, and probably not even then.

I would re-emphasize the point that too much ligation mix is inhibiting. Aim at 5% by volume of your cells. Even that is somewhat inhibitory in our experiments.

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Have you tryed Electroporation?? I use DH5 alpha and that allways works for me.

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