Team:Cambridge/Protocols/Electrocompetentcells

This protocol was taken from [http://openwetware.org/index.php?title=Electrocompetent_cells&action=edit Openwetware.org].

Growing Electrocompetent Cells

Originally from Sambrook and Russell's "Molecular Cloning: A Laboratory Manual" Third Edition.

(Note: once the cells are grown and have been placed in the first ice bath, you do not want the temperature of the sample to rise above 4 °C at any point. Therefore, many of the instructions are given with this in mind; always think ahead to the next step and to how you are going to keep your cells from warming up. This includes prechilling tubes and keeping all wash materials and samples on ice.)

Fundamental idea of protocol

The main idea here is to get a very HIGH DENSITY (hence the centrifugation), UNCONTAMINATED (hence all the autoclaving), culture of cells in a completely SALT FREE solution (this is to prevent arcing in the electroporation kit). Depending on what OD600 readings are taken, more centrifugation steps might be needed in smaller volumes of glycerol/GYT in the later stages of the protocol.

Materials

GYT (glycerol, yeast extract, tryptone)

• 10%(v/v) glycerol

• 0.125% (w/v) yeast extract

• 0.25% (w/v) tryptone

DI water
10% Glycerol

Autoclave beforehand

1x250ml flask (or use new plastic one)

2x 475ml LB in 1L flasks

1.2L DI water in 1L bottles (make more than this...)

4 x 250ml flat bottomed centrifuge tubes

GYT

10% Glycerol

(For the GYT and Glycerol, only small amounts are needed but it's always useful to have sterile stock so get some 1L bottles out)

Eppendorfs for the 40μl aliquots

Method

Important: All steps in this protocol should be carried out aseptically

• Inoculate:

Prepare flask containing 50 ml of LB medium. Pick up a single colony of cells from plate (using a sterile toothpick) and swirl around inside flask. Incubate the culture overnight at 37°C with vigorous aeration (250 rpm in a rotary shaker).

• Dilute and incubate:

Inoculate two aliquots of 475 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/min in a rotary shaker). Measure the OD-600 every twenty minutes (this step will take around 1.5-2 hrs).

• Rapidly cool culture:

Once the OD-600 of the culture reaches 0.5, rapidly transfer the flasks to the pre-made ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in the ice-water bath as well.

Note: After this point, do not let your cells warm up past 4°C

Note: When harvesting cells by decanting, be very careful to not disturb the pellet-- this could result in a much lower yield. If necessary, aspirate instead of decant the supernatant. Get someone to show you how to aspirate. Also, if the pellet seems loose, sometimes it is helpful to re-spin the cells down.

"Note: A standalone centrifuge is required during this protocol and the correct rotor must be selected"

• Centrifuge 1:

Transfer the cultures to 4 250ml ice-cold centrifuge bottles (in 200ml volumes - i.e. discard 200ml). Harvest the cells by centrifugation at 1000g (2500 rpm) for 15 minutes at 4°C. Decant the supernantant and resuspend the cell pellets in 200 ml of ice-cold DI water (200ml per centrifuge bottle i.e. a 1:1 suspension).

• Centrifuge 2 (water):

Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellets in 100 ml ice-cold DI water (per tube). If you like you can now combine two tubes into one - as long as the centrifuge is always balanced. You will now have 2 x 200ml suspensions to be centrifuged.

• Centrifuge 3 (water):

Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 10 ml ice-cold 10% glycerol.

• • To spin down your pellet in 10 ml, transfer your suspensions into 15-ml Falcon tubes instead of using the round-bottom tubes. A smaller centrifuge can now be used

• Centrifuge 4 (glycerol):

Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Carefully decant the supernatant and use a Pastteur pipette attached to a vacuum line to remove any remaining drops of buffer (A normal pipette will also do - DO NOT POUR the supernatant or you WILL lose culture).

• Resuspend in GYT:

Resuspend in 1 ml ice cold GYT. This is best done by gently swirling rather pipetting or vortexing and takes a long time - be patient.

• Measure OD:

Measure the OD-600 of a 1:100 dilution of the cell suspension. (In the cuvette, mix 0.99 mL water and 0.01 mL cell suspension). Note: The desired concentration is 2.5*10¹¹ cells per mL, giving 1*10¹⁰ cells per 40 μL. This corresponds to an OD-600 (after 100x dilution) of roughly 3.75. It is difficult to reach this value, but it is still important to know the concentration of cells to calculate efficiencies.

• Dilute the cell suspension to a concentration of 2 x 10^10 to 3 x 10^10 cells/ml (1.0 OD600 = approx. 2.5 x 10^8 cells/ml) with ice-cold GYT medium.

• Test for arcing:

Transfer 40 ul of the suspension to an ice-cold electroporation cuvette (0.1-0.2 cm gap, on middle shelf next to electroporator) and test whether arcing occurs when an electrical discharge is applied. Place the cuvette in the green holder attached to the machine. Go to option 4, Pre-set protocols; choose bacterial; choose the correct choice for your size cuvette, probably the first option for a .1 cm cuvette. If arcing occurs, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq).

• Storage:

Store cells at -80°C until they are required for use. For storage, dispense 40 ul aliquots of the cell suspension into sterile, ice-cold .5 ml microcentrifuge tubes, drop into a bath of liquid nitrogen (This rapid freezing is not strictly necessary but could be fun) and transfer to a -80°C freezer. To remove the tubes from the liquid nitrogen bath, bring out into the hall along with a storage box, and pour the tubes and liquid nitrogen into the box. Once all the tubes are out, close the box most of the way and let the liquid run out into the hallway. Try not to do this in the very center of the walkway!

To use frozen cells: Remove an appropriate number of aliquots of cells from the -80°C freezer. Thaw the tubes on ice.