Please help. I've been using the electroporation method of Kim et al., 2005 (J. Appl. Microbiol.) for the transformation of plasmid DNA to lactic acid bacteria.
My electroporation parameters are almost exactly similar to Kim et al. 2005:
Incubated in MRS with 1% (wt/vol) glycine
1 ul aliquot of 1 ug of plasmid DNA in 50 ul competent cell
10 kV, 200 ohms, 25 uF
Dissolve in MRS broth then incubate for 2 h at 37 C.
I've tested this protocol several times for weeks, and I know its working (usually I got 20-30 colonies) but for the past two weeks (I did 6 trials already), but its either I have 1-2 colonies per plate or nothing. Am I missing something? Is there some step or detail that I am not considering? Your opinion will be valuable, please help! Thanks in advance.
try this protocol:
inoculate 100ml of MRS+1%glycine+0.5M sucrose with O/N culture grown in the same medium
grow to OD600=0.2-0.7
wash 3x in ice-cold 0.5M sucrose+10%glycerol
resuspend in 1ml of ice-cold wash buffer
electroporate 40ul of cells with 200ng of DNA in ice cold cuvette
add 4ml of ice cold MRS+0.5M sucrose+2mM CaCl2 leave on ice for 5 min
recover 2h at regular growth temperature (induce Ery 50ng/ml if necessary)
concentrate by centrifugation to 0.5ml
plate on MRS+0.5M sucrose
sucrose is important from the very beginning to introduce isotonic conditions so glycine can impair cell wall synthesis, up to the end to keep cells intact
Other than sucrose, are there any other steps, handling, electroporation conditions, just anything that is being overlooked during electroporation? Sometimes, I think, I'm doing the same thing perfectly the same like in other trials. However, there might be some incremental difference in the method or handling that I am doing that I tend to overlook?
@ProfMarcinSchmidt Based on your experience, what could be those factors?
I think sucrose is very important, recovery step after electroporation, induction of antibiotic resistance with low conc of antibiotic. I never use any salts in electroporation buffer (I use GenePulser, Bio-Rad).
Be gently, do not vortex at any step, just gently tap bottom of tube to resuspend pellet (it takes some time so remember to cool tube on ice during resuspension), do not pippet too much, use cut pippette tips with wider openings.
ice need to be wet, in case you use ice from -20C this might be a cause
you may also have toxic gene in your plasmid - in this case you will need tightly controlled induction promoter
Thanks, Professor!
I am also wondering how long does it take to see colonies in the plates? 2 or 3 days? The protocol I was using also said that maximum is 3 days. However, sometimes I don't see good expression only after 3 days.
How do I know that the gene is toxic to my host? By the way, I will do electro-transformation tomorrow. I will consider all your suggestions above and let you know my results the end of this week.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning