Hello,
I am having difficulties working with E. coli WK6 cells.
I have used the WK6 cells to express Nanobodies for almost three years, and everything has gone well. One difficulty I encountered was preparing a fresh competent cells every month, unlike other E. coli strains in our lab.
The second difficulty I am facing is that I've tried six times over the past month to create a new batch of cells. Despite trying different approaches, I've been unable to get competent cells that are able to preform.
My protocol for wk6 preparation is based on Inoue et al.'s (1990) Gene 96:23-28.
I would appreciate any help you can offer.
How are you storing your WK6 strain and are you streaking out on minimal media to ensure retention of the F' episome?
I have a glycerol stock in the -80°C freezer. Before preparing a new batch of competent cells, I seed the cells from the glycerol stock on M9 minimal medium plates. Michael J. Benedik
If the colonies look normal then I can't think of any reason you are having problems. That strain is fairly standard with respect to its genotype.
It is true that different strains respond with differing efficiencies to various protocols for making competent cells. So you might have better luck with a different protocol, but I'm not certain.
The colonies seem normal to me. I've already tried different protocols, but I have yet to work so far. If I understand correctly, you have experience with this strain and haven't encountered any issues? Do you have other a protocol for preparing WK6 cells or any tips that you can share? Michael J. Benedik
I have never used WK6 but have used similar strains (JM101 or TK1) and never had any issues with them.
I wonder whether your strain stock is perhaps contaminated with M13?
We frequently just used the traditional CaCl2 method but also had good luck with the following: TSS Competent E. coli Transformation Chung, C. T. & Miller, R. H. (1993). [43] Preparation and storage of competent Escherichia coli cells. In Methods Enzymol, pp. 621-627.
I find it difficult to believe that my stock has been contaminated by M13. When I seed them in an M9 plate, the colonies appear normal, so how is this possible? Shouldn't I observe any changes in the morphology of the colonies if my cells were contaminated with M13? Michael J. Benedik
I don't have experience with those cells, but I have made competent cells before. Here are some pitfalls:
It is very easy to contaminate competent cells. DNA is hard to get rid of. Anything that is reusable can be contaminated with DNA, and the competent cells will soak it right up. When we made our own competent cells, we cleaned the glassware with soap, water, ethanol, water, nitric acid, purified water, then we autoclaved.
Also, competent cells are very easy to make no longer competent by temperature variation. If they are not performing, check whether there are any storage steps over -80 C (are they at the front of the freezer?) or any procedure steps over 4C (are you thawing them between your fingers?)
Competent cells are very finicky and don't like to be pipetted. Shear force can kill them. When aliquoting, use a large bore serological pipette. When using the cells, mix by gently flicking the tube no more than twice and then wait until they close up before shaking, vortexing, pipetting, etc.
Homemade cells are not as good as store bought cells in the sense that there will be a lower percentage of the cells that are competent and a higher percentage that are not competent. You might have to plate a lot more to get the same result.
I used the Inoue protocol at room temp, since I didn't have a cold incubator, growing cells for 12 hours, very inconvenient but worth it. Don't let them overgrow the O. D. specified.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes