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Team:Trieste/protocols
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The Jolly JoCare
a safe probiotic platform for protein expression
Week 1
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Preparation of Competent Cells
Work as sterile as possible.- 1. Take an 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated.
- 2. Grow the cells on a shaker at 37°C until they reach an O.D.600nm=0,6.
- 3. Transfer in sterile Falcon (50mL).
- 4. Centrifuge it at 4500 rpm for 10 minutes at 4°C.
- 5. Resuspend the bacteria pellet on ice in 0,1M of ice cold CaCl2.
- 6. Keep this suspension on ice for overnight.
- 7. Centrifuge it at 4500 rpm for 10 minutes at 4°C.
- 8. Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v).
- 9. Dispense in aliquots and freeze cells at -80°C.
Transformation - Heat Shock
Miniprep
Gel Extraction
Clean colony PCR
E.L.I.S.A.
Western blot
Stripping
