For many years, I have tried occasionally to make competent cell from E coli that was revived from competent cell. It never worked well, normally got super low efficiency, even I tried streaking the cell more than couple times and allowed the cell grew for several days before making competent cell. I could not find any protocol or paper addressing this problem. There is a couple discussions but contradicting. By the way, I normally do electroporation.
Any one having same problem? reason? literature?
Delin Liang .
You can try by chemical method i.e. CaCl2.
1. From very fresh LB streak plate, Pick a single colony and inoculate into appropriate media (50mL).
2.Grow overnight @ 37*C.
3. Transfer the overnight 5mL culture to 100mL media and grow for another 5-6 hrs.
4. pellets cells in chilled autoclave centrifuge tubes(5000rpm,10min,4*C)
5.Decant supenatant.
6. Resuspend in 1/4 vol of 0.1M Cacl2. Hold on ice for 5 min.
7. Centrifuge (4000rpm,10min,4*C)
8. Decant supenatant.
9. Resuspend in 1/20 vol of 0.1M Cacl2. Hold on ice for 20 min.
10. Centrifuge (4000rpm,10min,4*C).
11. Decant supenatant.
12. Resuspend in 1/100 vol of 85% 0.1M Cacl2 and 15% glycerol.
13. Pippet 100uL into each tube and place immediately on ice.
14. Store in -80.
You mean that the chemically competent cells you prepare from commercial batches of competent cells are not good but those you prepare from other sources are good? Or that the competent cells you prepare are never good, period?
If the latter, that's not surprising. Reagent quality and apparently minor tweaks to the protocols that are not easy to convey in written form (like the way you resuspend cells, etc) have an inordinate influence on the resulting transformation efficiency of home-made competent cells. There is a reason why life science companies can get away with the prices they charge for them. Try to get tips from someone with expertise on preparing high quality competent cells, or stick to electroporation.
Hope it helps!
I've recovered multiple strains from competent cell stocks (both electrically and chemically competent), all I did was take a loopful of the strain and struck it out on LB agar (with appropriate antibiotics if it contained a plasmid) and every time they grew within 16 hours at 37° or 24-48 hours at 30° for temperature sensitive strains. Then I just picked a single colony, inoculated it into LB broth, grew that overnight and made a permanent glycerol freezer stock of it, and made competent cells from subcultures of that freezer stock. If it's taking them several days to grow visible colonies something isn't right.
I'm guessing it's like Alejandro said and it's something wrong with your technique. Some errors I've either seen people do or have done myself with electrically competent cells:
- Letting cells grow too long, usually an OD600 of ~0.5 gets good results.
- Resuspending cells by vortexing instead of pipetting. You can usually get away with this resuspending them for the first glycerol wash but anything after that they become a lot more fragile to shear forces.
- Not making the cells dense enough before freezing down aliquots. This I think is the hardest to explain and I've never measured it but the cell suspension should be totally opaque, like milk. A 200 mL of a culture with an OD600 of 0.5-0.6 only makes about a dozen 50 µL aliquots of competent cells for me.
- Not resuspending the cells homogenously for the glycerol washes. If you have any chunks they're not going to be washed very well.
- Using tap water to make reagents/media instead of deionized or double distilled water.
- Centrifuging cells too hard between washes, I wouldn't subject them to anything above 5000 rcf, 3000 rcf for 20 minutes in a 50 mL conical usually works very well for me.
Thank you for the helps, especially for Alexandra Johnson . They are helpful. One thing I want to argue is that I made a lot of competent cells successfully before and now. I know most of the tricks.
But I have never succeeded in making competent cell from E coli that is revived from competent cell. This is important for me because I do a lot of transformations. I do not want to keep buying, especially some not public available strains. My feeling is that competent cells have gone some changes and are not good for making competent cell again. It good to know that Alexandra have succeeded in this but my case seems different.
By the way, I only buy electroporation competent cells and make electroporation competent cells by myself.
Oh, you meant electrocompetent cells. Sorry, my bad. No, I've never noticed what you are describing.
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