I have some problems with electroporation of Bacillus subtilis WB600.I need help please
1).How long does it take for Bacillus subtilis to grow positive colonies overnight after transformation? Generally, it takes 16 hours to grow a "small white dot", is it true?
2).Can Bacillus subtilis be identified by colony PCR? I pick up the dot and drop it into 10ul water, heat at 95 ℃ for 10 minutes, then - 20 ℃ for 5 minutes, take 1ul as template, but there were no bands in the results.
3).How much is the final coating amount for Bacillus subtilis transformation? I take 100ul from 1ml recovery system,. But after overnight cultivation, a layer of membrane which seems like coarse sand grows on the surface of the plate. Is this normal?
Thanks for your advice
I have worked with Bacillus subtilis 168 strain. However, basic handling techniques are same for most of Bacillus subtilis strains. My experience might be helpful for successful transformation and screening.
The recovery time after transformation varies case by case. It is best to consider the colonies that appear within 48 hours for screening.
Good quality and consistent colony PCR of Bacillus subtilis clones is possible. I have used following method for colony PCR:
Plating volume vary based on efficiency of transformation. You can try serial dilution to optimize the volume that is best for your experiment. I usually centrifuge whole 1 ml of the recovered culture, discard 900 uL, mix the pellet in remaining recovery media and spread on a selective agar plate.
Best wishes for your research.
Dear Zhang,
The growth time of any bacteria increases after electroporation. It is best to incubate the culture for 48-72 hrs after electroporation.
Bacillus having a tough cell wall, colony PCR is not recommended, since it is too hard to lyse by physical means, even sonication also requires a bit optimization. I work with Lactobacillus, and according to me, for better and undsputed results, it is better to go through the general protocol i.e. DNA isolation by a quick protocol and then PCR.
100ul from 1ml is good enough to spread after electroporation. I would suggest you to spread in a serial manner i.e. 50ul, 100ul, 200ul. and observe the plates.
Thanks @ Anaya Pokhrel and @ Kazi Tawsif Ahmed for your suggestions. I think the main problem is the tough cell wall, so I did some change in my PCR protocol and finally got some positive results(16/23). If anyone else need help, you can have a try. Here is my protocol :
1. Pick a colony with a pipette tip and mix with 30 uL sterile water.
2. Heat at 95℃ for 15min
3. Freeze at -20℃ for 5min
4. Heat at 95℃ for another 5min
5. Centrifuge at 13000xg for 1 min
6. Take 1 uL of supernatant as template (10ul total volume)
The sandlike membrane seems like thousands of small colonies, maybe the concentration of chloramphenicol is not high enough(10ug/ml)
Thanks again for your help
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