Editing Electrocompetent Cells

== Procedure (for 2,250 ml cultures)<br>  ==

#Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm. 
#Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks. 
#Shake at 37°C and 250 rpm until the cultures reach an OD600 of '''0.4-0.7'''. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of '''10% glycerol''' on ice well in advance of harvesting cells 
#Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles. 
#Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth. 
#Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down. 
#Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells as before. 
#Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down. 
#At this point you can electroporate or freeze the cells away. To freeze, Add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen transfer them to a -80°C freezer. The cultures should be good for &gt;6 months.

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== Procedure (for 1 L cultures) ==

<br>'''Day 1'''<br>1. Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB<br>plate (no antibiotics). Grow plate overnight at 37°C.

'''Day 2'''<br>1. Autoclave:<br>2 L of ddH2O<br>100 mL of 10% v/v glycerol (molecular biology grade)<br>1 L LB (or your preferred media)<br>4 centrifuge bottles and caps<br>Lots of microfuge tubes

2. Chill overnight at 4°C: <br>ddH2O<br>10% glycerol<br>Centrifuge rotor

3. Prepare starter culture of cells<br>Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or your preferred media). Grow culture at 37°C in shaker overnight.<br>Notes:<br>You can also substitute other media like SOB, 2xYT, etc for LB if preferred. All glassware should be detergent free. Trace detergent residue reduces competency.

'''Day 3'''<br>1. Inoculate 1 L of LB media with 10 mL starter culture and grow in 37°C shaker. Measure the OD600 every hour, then every 15-20 minutes when the OD gets above 0.2.

2. When the OD600 reaches 0.35-0.4, immediately put the cells on ice. Chill the culture for 20-30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time.

IMPORTANT NOTES:<br>• It is important not to let the OD get any higher than 0.4. The OD should be carefully monitored and checked often, especially when it gets above 0.2, as the cells grow exponentially. It usually takes about 3 hours to reach an OD of 0.35 when using a 10 mL starter culture.<br>• It is also very important to keep the cells at 4°C for the remainder of the procedure. The cells, and any bottles or solutions that they come in contact with, must be pre-chilled to 4°C.

3. (Spin #1) Split the 1 L culture into four parts by pouring about 250 mL into ice cold centrifuge bottles. Harvest the cells by centrifugation at 1000 g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C.

4. Decant the supernatant and resuspend each pellet in 200 mL of ice cold ddH2O.

<br>5. (Spin #2) Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C.

<br>6. Decant the supernatant and resuspend each pellet in 100 mL of ice cold ddH2O.

<br>7. (Spin #3) Combine resuspensions into 2 centrifuge bottles (so each contains about 200 mL of cell suspension). Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C. At this step, rinse two 50 mL conical tubes with ddH2O and chill on ice.

<br>8. Decant the supernatant and resuspend each pellet in 40 mL of ice cold 10% glycerol. Transfer each suspension to a 50 mL conical tube.

<br>9. Harvest the cells by centrifugation at 1000 g (~2100 rpm in the Beckman GH-3.8 rotor) for 20 minutes at 4°C. Start putting 1.5 mL microfuge tubes on ice if not already chilled.

<br>10. Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice cold 10% glycerol by gently swirling. The final OD600 of the resuspended cells should be ~ 200-250.<br>

11. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cell in the -80°C frreezer.<br>

== Electroporation Protocol  ==

#Turn on electroporator and set to 2.0 kV, 200 ohms and 25 µF.
#Place recovery SOC in 37°C water bath.
#Pre-warm LB-antibiotic plates at 37°C.
#Thaw cells on ice for 10 min.
#Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
#Flick the tube containing cells a few times to mix and add 50 µl to the microcentrifuge tubes.
#Add 1-3 µl of DNA solution (in DI water) to the cells in the microcentrifuge tube.
#Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse.<br>
#Immediately add 950 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
#Rotate in the 37°C incubator for 1 h.
#Make appropriate dilutions in SOC and plate.
#Incubate at 37°C for 18 h.<br>

== <br> Electroporation 2<br> ==

#The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.
#Turn on electroporator and set to '''1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.'''
#Place recovery SOC in 37°C water bath.
#Pre-warm LB-antibiotic plates at 37°C.
#Thaw cells on ice for 10 min or use freshly made cells.
#Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
#Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.
#Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.
#Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).
#Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
#Rotate in the 37°C incubator for 1 h.
#Make appropriate dilutions. When using 10 pg of DNA, make two dilutions:
#Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)
#Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
#Incubate overnight at 37°C.

Calculation: 

If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml) 

cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19) <br> 

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[[Category:Protocols]]

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