protocol for electrocompetent cells preparation - (Feb/06/2006 )
Hi! Does anyone have a protocol for preparing electrocompetent coli cells in a small scale? I mean, just a couple of tubes. I´ve looked it up in manuals and internet, but only found protocols that use liters of media, etc. Get the idea? Thanks!!
I haven't done this myself, but isn't it possible to just scale these protocols down to the size you want/need?
this is possible to adapt quantities, but it's not suitable for solutions <100ml...You need to do a preculture and then dilute at least 20 folds.
ger225 - for electroporation into e coli (JM107, DH5a, etc) I used to always use a small-scale protocol to get the best results
1. grow up a small overnight
2. in the morning, inoculate a 1:20 volume (I used to put 1 ml O/N into 19mL)
3. grow to low OD / early log phase. do not grow past early log phase.
FROM HERE ON, DO IT ALL ON ICE
4. transfer 1.5 mL to chilled microtubes
5. spin on high 3-4' in 4C microfuge
6. wash with ~500ul sterile ice-cold distilled water (resuspend by pipetting up and down until it is thoroughly mixed)
7. repeat wash step 3 to 5 times
8. resuspend in a final volume of 200ul sterile ice-cold H2O
this gives you 4 electroporations / tube, if you use 50 ul volumes for your electroporation
add your drop-dialyzed (or otherwise desalted) DNA; 2 or 3 ul seems to be best but you can push it to 5 ul if you expect a lower yield of transformants
electroporate as usual, with appropriate positive and negative controls to assess competency and cell viability
I do not have a reference, unfortunately; it was something my mentor-at-the-time showed me that worked well. if you try to wash less than 3 times, there will still be remaining salts from the media that will give you a 'pop'. I always washed 5 times unless I was in a really big hurry, it only takes a minute. If you notice the pellet is getting a little soft, you can increase spin time by a minute.
oh, PS, you cannot freeze these and use them later, there is no cryopreservative. just toss what you don't need
Thanx aimikins, the protocol works fine! I made some changes, though. 1 ml ON culture to 19 ml is way too much for me. I add 100 ul to 10 ml. Also, I tried to increase the numbers of cells to be electroporated. So, I poured all of the 10 ml to a 15 ml Falcon. Then, I pelleted the cells, resuspended in water an transfered them to an eppie. Then, I washed 4 times. It worked great. Recently, I resuspended in the end in 10% glycerol so that the cells could be storaged. I haven´t use them yet though...
hey, I'm glad it worked for you
I gave up electroproration a long time ago...those cuvettes are a PIA...now I do a heat-shock with chemically competent cells, I find it to be easier overall and I like to make the cells up in batches. But it is always good to know more than one method
let me know if the glycerol-preserved cells work...I'm thinking maybe that much glycerol is not the best if you want to electroporate...but I don't remember for sure and I've never tried it.