Hi,
I'd appreciate some advice on a recurring issue of mine. which is that when I'm transforming chemically competent BL21 cells I am obtaining very few (if any colonies) after static 37 degrees overnight incubation on LB/kan plates. The plasmid contains the kanR gene.
The protocol I have been following is as follows:
Add 2 µl of plasmid (100-500ng) into BL21 cells.
Incubate on ice for 30 mins
Heat shock for 90 sec at 42 °C
Place on ice for 2 min.
Add 250 µl LB and incubate at 37 °C with shaking for 1hr.
Plate out culture onto LB-Kan/ LB-Amp agar plates. Incubate at 37°C overnight.
Pick 5 colonies into LB-Kan/ LB-Amp and incubate overnight at 37 °C.
If anyone would lime further detail please let me know and I'll do my best to provide. looking at other protocols, I am concerned I may not be mixing the plasmid with the cells enough initially, and other protocols often have shorter heat shock periods.
Thanks in advance,
Tom
Your heat shock is quite long, I would limit it to 30 or 45 seconds. However I doubt that is the primary problem.
Aside from that the protocol seems fine. So if you are getting very few colonies it is either the plasmid DNA or the competent cells.
Double check that your plasmid is actually good (or try a different Kan-R plasmid).
Confirm with a control plasmid that your competent cells are good.
Hi Michael J. Benedik
Thanks very much for your response. I will amend my protocol so the heat shock period is shorter.
I am fairly confident in the competency of the BL21 cells; previously I transformed an aliquot with a colleagues ligation which we know works cell and many colonies were obtained.
With regards to the plasmid, how might I check it's quality? I am a new researcher so not sure how I'd go about this.
Thanks,
Tom
Just run a bit of your plasmid uncut on a gel and see if you have a nice plasmid band.
Are you getting no colonies with pure plasmid DNA or is this a ligation from a cloning? I had assumed from your first post it was just pure DNA but if it is a cloning then there are many other possible places where it went wrong.
Michael J. Benedik it is a cloning construct, but I didn't do the cloning. The process has been a bit long winded (and I am completely aware that this process is not ideal) but as I am a new PhD student my supervisor wanted me to follow this procedure so I get experience with cloning, minipreps etc. What happened is this:
1. Streak transformed BL21 cells (containing construct) from glycerol stock on LB/kan.
2. Overnight and miniprep from BL21 cells. Transform plasmid into Top10. (This was successful).
3. Overnight and miniprep to transform back into BL21.
I think the point is that I will have my own stocks that I made up myself. But the construct is okay since it was used in previous research project.
If it is pure plasmid DNA then the issue is pretty much either the competent cells or the plasmid. Run some uncut plasmid on a gel to be sure it looks good. Or borrow some other plasmids to use as a control.
Did you make the competent BL21 yourself or is it commercial?
Again the point here is do all the controls so you can figure out where things are going wrong.
Hello Tom. Are these competent cells prepared in the lab or bought commercially? If others in your lab are having no issues with the cells then it is likely your protocol or the plasmid. Isolating plasmid from BL21 cells is risky business because, unlike DH5a cells, BL21 cells are not meant to maintain plasmids (mutations can arise). Are you preparing your own LB agar plates using your own antibiotic stocks? If so, is it possible that your antibiotic concentrations are off? Another possibility may be that you are killing the cells when plating them. If you use a glass “hockey stick” that is drenched in alcohol and then flame sterilized prior to spreading the cells. You may not be cooling off the hockey stick enough prior to spreading the cells. Good luck!
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