I cloned my gene of interest in pET-22b vector. I could transform DH5α E. coli strain. But I am not being able to transform expression strain of E. coli. My transformation protocol is; thaw the competent cells for 30 mins in ice after adding vector, heat shock for 45 sec at 420C, place back in ice for 2 mins, add 500 µL SOC, incubate at 370C and then transfer to LB plate. And I am using ampicillin at concentration of 150 µg/ml. So, why do I get bacterial colonies for DH5α but not for other strains?
Hi Rakesh, DH5alpha are really optimized for plasmid transfomations, so you should expect loads of colonies from chemically compentent cells (i.e. >1000 colonies for 10 ng of plasmid using chemically competent cells). Other strains such as BL21 can be less efficiently transformed and will yield less transformants - make sure you boost your transformation skills to this high efficiency for DH5alpha before you attempt other strains.
150 ug/mL ampicillin seems to be excessive - 50 ug/mL generally are sufficient.
Good luck!
Hi,
Here are some suggestions for improving transformation:
Use freshly prepared competent cells. Thaw competent cells on Ice.
After Transformation, grow cells for 1 hr at 37°C without antibiotic, spin down the culture and re suspend cell pellet in 100uL and plate.
Use 50 -100ug/mL Antibiotic on Plate.
You should check your competent cells with a control plasmid to be sure they are still good. The most likely explanation is that they are not. The ampicillin concentration should not matter, we have used from 50 to 500 without problem. So the cells are most likely the problem assuming you know that your plasmid is good.
I agree with Michael J. Benedik's opinion. Please check the competent cells using the control plasmid. Maybe you can also try the electroporation.
It may be helpful to read the book at the link.
Book Microbial Genetics Questioned to Understand
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