I prepared the competent based on the protocal.
I have plotted the growth curve of the host strain B.subtilis and add 0.5ml of culture of SPI (OD660=1.8) which was incubated for about 4.5h into SPII. I incubated the culture for 1.5h, andded the 100ml 100mM EGTA, and then divide it among 14 ml round- bottomed test tubs, dispensing 300ul into each.
then I added 1ug DNA into the competent and incubated it at 30℃， 100rpm.
I did this experiment for several days but there is no colony growing on the plate.
I do not know the size of your plasmid, but suggest that use a small plasmid, 5-6 kbp, as a control to find if there is something wrong with your competent cells. If control plasmid grew then you can be sure that your competent cells working well. Now you can use ULTRA COMPETENT CELLS for your plasmid, which allow you to transform large fragments much more easier.
In all the steps ensure to use correct antibiotic compatible with your plasmid.
Some question to know about your experiments:
Replication origen is for Bacillus? or plasmid has recombination sites for Chromosome?, Plasmid come from RecA+ E. coli? Do you have some controls positive or negative?
Hi,
I use CalC2 method for competent cell preparation followed by heat shock transformation at 42 C for 90 sec. Then incubate it for at least one hour at 37 C and select them on selection plates for over night.
Hi， Hammad Ismail，
Did you use heat shock in the transformation in B.subtilis?
Hi,Juan Campos-Guillén,
I mad the plasmid with the target gene in E.coli, and then transform it into the B.sutilis.
Acturally, I transformed the plasmid into B.subtilis successfully, but the yield is not very good (about 68 colonies per 1ug plasmid). I use the traditional SPI and SPII as medium. What can I do to improve the yield? my plasmid is about 6kbp. Thank you.
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