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Category: Experimental Procedures
Author: Admin eLabJournal

Labels: Transformation

Materials
 

- SOB medium  
- Antibiotics  
- 10 % glycerol  
- shaking incubator  
- centrifuge
-spectrophotometer
 

Experiment settings

- Strain name:    
- Culture volume: ml   
- Antibiotics:  
- Growth temperature: °C   

Step 1
Inoculate ml  of SOB medium with ml  of E. coli    overnight culture
Step 2
Add antibiotics only if required: Add µl    to the inoculated SOB medium
Step 3
Grow the cells for approximately 2-3 hrs at °C  while shaking at 220 rpm until the OD600 is between 0.4-0.5
Step 4
Spin down the cells at 4,000 rpm for 5 min at 4 °C. In all next steps: keep cells on ice
Step 5
Remove the supernatant and resuspend the pellet in 10 ml ice-cold 10% glycerol
Step 6
Collect the cells by centrifugation at 4,000 rpm for 5 min at 4 °C.
Step 7
Repeat step 5 by removing the supernatant and resuspending the pellet in 10 ml ice-cold 10% glycerol
Step 8
Spin at 4000 rpm for 5 min and resuspend the final pellet in µl  10% glycerol
Step 9
Make alliquots of 100 µl and store at -80ºC or proceed to  electrotransformation of E.coli