Electro-competent cells. How do I make them "more" competent? - Need advice on increasing the efficiency of Top 10 E. coli (Nov/07/2007 )
I've been trying to make Electro-competent Top 10 E. coli for a couple of weeks. I've followed 3 different protocols. I've been gentle with the cells. I've kept them as cold as possible. I've flash frozen them after the final aliquot in dry-ice and ethanol and I've just quickly aliquot them and then placed in a minus 80 freezer. No matter what I do, I can't seem to get efficiencies greater than 10^8. On the last trial, they did test out to 10^9 but just barely. The professor that I'm interning with said that a person should be able to make 10^10 or higher electro-competent cells in their sleep. It's really a problem with my ligation transformations as none of them are working and we've ruled out the other possible causes. Any suggestions? Other than me lacking "golden hands" (as one of the grad students in the lab has put it) I'm not sure what I'm doing wrong.
What strain are you using? The competence is highly dependent on the strain. Do you have positive experience in transforming cells made by others? Your transformation technique, rather than your cells may be the problem. Are you transforming with other than ampicillin resistance? We've found that outgrowth for 1.5 to 2 hours works much better than 1 hour.
I've been using portions of a frozen stock of chemically competent top 10 cells as my seed. That stock was made by the previous undergrad intern from commercial top 10 cells, sorry I don't know which brand. Her stock tested out at 10^7. If I shouldn't be using these as cells, what strain should I be using?
Try to compare efficiencies with the growth rate. OD the bacteria and try out different ranges. This may help.
Good Luck !!!
Top10 (all from Invitrogen) should work well as a strain. I'd buy some commercial competent cells just as a control to make sure my technique and electroporator were working well. What cuvettes and voltages do you use? How are you treating the cells after electroporation?
I'm using .1 cm cuvette on the standard setting of 1.8 kV. I immediately add 1ml of LB with no selection. I will transfer the sample to an eppie and place a the 37 C shaker for an hour if they are Cm resistant. If they are Amp, then I'll usually just plate right away. I talked with one of the grad students. I'd been resuspending them by gently swirling in an ice-water bath. This would take awhile, sometimes 15 minutes. She resuspends with the pipette and is done in a minute or less. I switched method's half way into making new cells and saw no improvement in efficiency. I in the process of making a new batch using her method. Any other suggestions?
1,8 KV sounds high for a 1mm cuvette. I would either switch to 2mm cuvettes and keep that voltage, or lower the voltage to 1 - 1.2 KV. 2mm cuvettes are much easier to deal with -- the pipet tip fits into them more easily. I find that "washing" the cuvette by pipetting in LB then pipetting it out (repeat) works well in emptying the cuvette. Acting fast is important. I alway let the cells grow without selection, even with amp resistant strains. I find that efficiency improves substantially the tet and cm selection when extending the non-selective growth to 1.5 hours.
I used the grad student's method, focusing on minimizing the about of time they were out of the cold centrifuge rather than on being gentle and my results were 2.0 x 10^10 on a 1/10,000 plate. I needed 9 colonies on that plate to make them 10^10, still a noticeable improvement for me. I'll switch to large cuvettes, a lower voltage than the standard setting for the BioRad MicroPulser, and giving the samples the hour long rebound. Thank you all for advice. It has helped.
I have done electoporations for two different kind of E. coli strains. With the first strain I got a lot of colonies after the transformation (by recombination). However, with the second strain I did not always get a lot of colonies. I was told that at the beginning of the project they got a lot of colonies with that strain, but somehow the recombination efficiency was lost? I have prepared the electrocompetent cells the same way for both strains. The only different thing is that I now use a different electroporator. The electroporator I am using now is the Biorad MicroPulser. I am using .2 cm cuvettes, program Ec2. The time constant I got varied from 4.5 to 6.0 ms (I know the time constant should be between 4.5 and 5.5 ms), but the actual volts did not change (2.5 kV). I do not know the brand of the previous electroporator; I only know the settings I used: 2.5 kV, 25 uF and 200 ohms. Does anyone know whether the settings correspond? I wonder how I can improve the transformation efficiency.
To Dinirich: I actually add SOC medium after the transformation. Maybe that can help improve your transformation efficiency. Besides, if you incubate the cells in Eppendorfs after the transformation, you could increase the incubation time to 2 hours or you can incubate them in a larger culture tube, which can increase the amount of aeration. And I do resuspend the cell pellets by pipetting. I wonder whether you can vortex them. And if you keep your cells too long in distilled water, will they burst due to the osmotic pressure?
Whoa here. I always calculate transformation efficiency using a an aliquot of supercoiled DNA with known concentration from a supplier (e.g. Invitrogen). Efficiency will always always ALWAYS be lower when using a ligation mixture because only a portion of the molecules in the reaction will be circular. Are you calculating efficiency from your ligation transformation or from a positive control?
Ginger