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From 2010.igem.org

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• Why Pichia
  • Pichia Vs Saccharomyces
  • Easy and Inexpensive to Culture
  • High production of foreign Protein while low levels of endogenous protein
  • Pichia pastoris versus Esherichia coli
• Protocols
  • Preparation Of Electrocompetent Cells
  • Preparation of Heat Shock Competent Cells
  • Transformation Using Electroporation
  • Transformation Using Heat Shock
• The Team
• Notebook

Contents 1 Preparation of Electrocompetent cells 2 Preparation of Heat Shock Competent Cells 3 Transformation of DNA using Electroporation 4 Transformation Using Heat Shock

Preparation of Electrocompetent cells

1. Inoculate 500ml of L-broth with 1/100 volume of a fresh overnight E.coli culture

2. Grow the cells at 37°C on shaker to an OD600.(dilute culture if desired OD is exceeded.

a. Best results are obtained with cells harvested at early to mid log phase so the desired OD may vary based on specific strain growth conditions

3. Chill cells on Ice for approximately 20 minutes. Keep cells on Ice for all subsequent steps in procedure and pre-chill all tubes before adding cells. If possible, centrifuge at 4°C.

4. Transfer cells to chilled 50 ml Falcon tubes and centrifuge at 4000x g for 15 minutes

5. Pour off and discard supernatant. Resuspend pellet in 50ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.

6. Resuspend pellet in 25 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.

7. Resuspend Pellet in 20 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.

8. Resuspend cell pellet in a final volume of 2ml of ice-cold 10% glycerol. Cell concentration should be about 1-3 x 1010 cells/ml

9. Make aliquots of 100µl and store at -80°C.

Preparation of Heat Shock Competent Cells

Preparation of Seed Stock 1. Streak TOP 10 cells on an SOB plate and grow for single colonies at room temperature 2. Pick single colonies into 2mL of SOB medium and shake overnight at room temperature 3. Add 15% glycerol 4. Aliquot into 1mL samples 5. Place in -80°C Preparing competent cells 1. Prechill 2mL centrifuge tubes 2. Inoculate 250mL of SOB medium with 1mL vial of seed stock and grow at 20°C to an OD600nm of 0.3 3. Aim for a lower OD not higher if possible for 16 hours at room temperature should work 4. Centrifuge at 3000g at 4°C for 10 minutes 5. Pellets should be resuspended in 80mL of ice cold ccMB80 buffer 6. Incubate on ice for 20 minutes 7. Centrifuge again at 4°C and resuspend in 10mL of ice cold ccMB80 buffer 8. Test the OD of a mixture of 200uL of SOC and 50uL of the resuspended cells 9. Add chilled ccMB80 to yield a final OD of 1.0 – 1.5 10. Incubate on ice for twenty minutes 11. Aliquot into chilled 2mL chilled centrifuge tubes 12. Store at – 80°C

Transformation of DNA using Electroporation

1. Thaw DNA and cells 2. Add 40uL of cells in a fresh microcentrifuge tube along with 1uL of DNA 3. After gently mixing transfer the contents of the microcentrifuge tube into an electroporation cuvette 4. Have the micropulsor set to Eco1 5. Hit the pulse button and hold it until you hear it beep 6. Immediately add 1mL of SOC broth to the electroporated cells 7. Transfer the contents of the cuvette to a micro centrifuge tube and inoculate the tubes at 37°C for an hour 8. Plate 20uL and 100uL on selective media plates 9. Also plate 100uL on regular media plates as a control to test the viability of the shocked cells 10. Inoculate these plates overnight for 14-16hrs at 37°C

Transformation Using Heat Shock

1. Thaw the cells and DNA on ice 2. In a microcentrifuge tube at 50uL of competent cells with 2uL of DNA 3. Let it sit on ice for 30minutes 4. Heat shock at 42°C for 60seconds 5. Let it sit on ice for 5minutes 6. Add 200uL of SOC broth 7. Incubate at 37°C for 2hours 8. Plate 20uL and 100uL on Selective media 9. Plate 100uL on normal media as a control to test viability of the heat shock cells