Editing Electrocompetent Cells (section)

== <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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