After electroporation when I plated the mixture on to medium plus antibiotic I got the colonies after 10 days,,i selected some colonies and performed a pCR to check the presence of recombinant plasmid (insert DNA and vector), I got 2 colonies positive. When I purified them again on antibiotic containing medium plate, it took two overnights to grow the colonies, that to very small in size. Please suggest how I ca I improve electroporation step, and get true transformants?
10 days - I would have thrown the plates away before that! Was that a typo?
Check all the small details - is the antibiotic correct, are you using the recommended amount in your plates, are your cells competent cells for electroporation, is your plasmid intact, do you accurately know the concentration of your plasmid, is there anything unusual about the plasmid (toxic, very large, etc), are you recovering cells for enough time after electroporation before plating?
Do you have positive and negative controls for electroporation for the same antibiotic plates? If the positive and negative controls for electroporation work fine, then perhaps double check your plasmid concentration and maybe do transformation at a few different concentrations.
Dear Amanda,
Thank you for your suggestion!
I had not incubated the plates for 10 days deliberately , I was sure that something had gone wrong and I would have to repeat the electroporation, so I just didnt care to remove the plate from Incubator before going for new year holidays. After coming back I checked the plates and found colonies.
I think the antibiotic is correct becoz I used antibiotic -plate as control. The plasmid with construct is 3.7 KB, and the concentration was 450ng/ul(I used 4uL of it for 100uL comp cells). Yes, I recovered the cells for 4hrs in EP broth after electroporation before plating.
I will keep your points in mind when I repeat the experiment.
Thank you very much!
1. You did not mention the amount (concentration) of antibiotic you added into the plate. Once, we had a culture, it took forever to grow (E. coli, 37oC). After we repeated the experiment and re-calculated the amount of antibiotic we added, it grew well again. So, check the concentration of your antibiotic stock (if you have one).
2. Also, check the temperature of your incubator. I remember that one time a RG member was seeking help for the failed growth of his Agrobacterium plate. Eventually, he found out that the temperature of the incubator has been changed from 28oC to 37oC.
By the way, what kind of bacterial competent cells were you using?
Dear Yau,
thank you for your input!
1, I am using chloramphenicol, 10ug/ml
2, Temperature of incubator is fine, bcoz its digital display.
i am using staphylococcus aureus competent cells.
Hi Madhuri,
Thanks for your response:
1. 10ug/mL, yes, I know, this is the 'goal', the final concentration. What I meant is to double check whether any human-error caused concentration change. For example, wrong stock concentration or add too much.
2. In our lab, even the incubator has digital display, we put an old-style thermometer in it. Just want to see whether the temperature inside is really what the 'digital display' says.
Good luck on your research,
Ah - now the 10 days makes sense! I have also left experiments when I'm mad at them.
Your chloramphenicol concentration actually seems low - 100 ug/ml seems standard.
Also, I have certainly had digital displays on heating blocks, incubators etc that are wrong - it never hurts to throw a thermometer into your incubator as a double check. However, if it was really off, I bet you would have noticed!
I have never used Staph aureus - I've only used E coli. From a quick search it looks like Staph aureus can be tricky. (http://mbio.asm.org/content/3/2/e00277-11.full Transforming the untransformable - eek!) If you don't get helpful responses, I'd suggest reposting specifically for problems transforming Staph aureus.
Please also refer to a similar discussion at RG:
https://www.researchgate.net/post/I_have_been_trying_to_electroporate_Staphylococcus_aureus_cells_for_a_while_now_without_success_Please_advise_Thank_you/2
E.coli DH5 aplha cells have very good transformation efficiency. Why dont you try that.
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