Preparation of electrocompetent E.coli cells
(Alonso-Stepanova Lab protocol)
There are numerous protocols online and in various methods books. The protocol we use is one of the simplest. The only trick is to do ALL of the washes and aliquoting in the cold (in a 4ºC coldroom or on ice). The idea behind the extensive washing is to wash away all of the salts from the media the cells were grown in. If any salts remain, the cells will pop upon electroporation. Glycerol is added in the final wash steps to help pellet the cells, as well as to be able to safely freeze the cells. The reason to keep the cells cold at all times is to increase their survival rate and, therefore, their electrocompetence, i.e. the transformation efficiency. Once in DI water, the cells become very sensitive to temperature changes and the transformation efficiency drops dramatically if cells are allowed to warm up above 4ºC at any step.
– Streak DH5alpha cells or equivalent on LB plates (10g tryptone, 5g yeast extract, 10g NaCl, 15g agar per liter) and grow overnight at 37ºC
– Inoculate a freshly grown single colony into 3-5ml liquid LB (same recipe, but no agar) and shake the culture overnight at 37ºC
– In the morning, dilute the fresh overnight culture 1:100 – 1:500 into 0.5-1 liter of liquid LB and grow for 3-6 hours until the culture reaches OD of ~O.5. Do not overgrow the cells!
– Cool the culture on ice for 10-15 minutes, transfer to pre-chilled 500ml Nalgene centrifuge bottles.
– Spin the cells in JA-10 or equivalent at 5000 rpm at 4ºC for 5-10 minutes to harvest the cells.
– Resuspend the pellet in 1 volume of ice-cold sterile DI water (gentle shaking on ice or in the coldroom is the best way to go).
– Spin the cells in JA-10 or equivalent at 5000 rpm at 4ºC for 5-10 minutes to harvest the cells.
– Wash the cells again by resuspending them in 0.5 volume of ice-cold sterile DI water.
– Spin the cells in JA-10 or equivalent at 5000 rpm at 4ºC for 10-15 minutes to harvest the cells (after washing, the cells become harder and harder to pellet down).
– Wash the cells with ~20ml [per liter of the original cell culture] of ice-cold sterile 10% glycerol and transfer the cell suspension to a pre-chilled 38ml Nalgene centriguge tube.
– Spin the cells in JA-20 or equivalent at 10000 rpm at 4ºC for 10-15 minutes to harvest the cells.
– Resuspend the cells in 3-4ml of ice-cold sterile 10% glycerol per liter of the original cell culture.
– Aliquot the cells into individual pre-chilled microfuge tubes (40 ul per transformation in a 0.1-0.2 cm gap electroporation cuvette). The cells are now ready to use. For long-term storage, freeze the tubes with 40 ul aliquots in liquid nitrogen and then place them into a -80ºC freezer. Dry ice plus EtOH works best for cell freezing in terms of retaining their viability and electrocompetence, but it is messier than liquid nitrogen. If using dry-ice, make sure no EtOH enters the tubes as it will immediately kill the cells!
-To use the frozen cells, thaw on ice the individual aliquots, add salt-free DNA to them (typically 1-5 ul), mix by pipetting, and transfer the mix to a pre-chilled electroporation cuvette. Electroporate the cells according to manufacturer’s instructions. Immediately after the pulse, add 1ml of room temperature liquid LB or SOC media to the cells, and recover them by shaking the samples at 37ºC for 1h. Plate the cells on LB plates supplemented with an appropriate selective agent, allow the excess liquid to dry/absorb into the plate. Incubate the plate up-side-down overnight at 37ºC.
Note: This protocol works both for E. coli and for Agrobacterium tumefaciens. For the latter, use 28ºC to grow the cell culture and to recover the cells after transformation. Longer growth time will be required to achieve desired cell density. Similarly, after transformation, plates will need to be incubated at 28ºC for 2-3 days for the transformants to grow.