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Team:Washington/Protocols/PUR Assay
From 2012.igem.org
PUR Esterase Assay
- Transformed MG1655 with PUR Esterase pGA3K3 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K892012 BBa_K892012]
- Followed the [https://2012.igem.org/Team:Washington/Protocols/Elect. electroporation protocol]
- Plated on 200µL of rescue on LB + 1% kanamycin agar plate
- Plated MG1655 on LB agar plates
- Next day, picked 1 colony from each plate and grew them in 50mL of TB
- Added kanamycin to the the transformed cell's liquid culture (final concentration of 1% mass/volume)
- Next day, spun the 50mL overnight cultures down at 4000g for 10 minutes
- Poured out the supernatant
- Resuspended cells in DI water to make each culture equal in 1-cm path length OD (we chose and OD of 1.4)
- Aliquoted out 1mL from each culture into 3 different microcentrifuge tubes tubes.
- Using a sonicator, sonicated the aliquots at an amplitude of 20 with 1 second pulses on and off for 30 seconds.
- Applied lysate to pre weighed samples of foam within 25mL culture tubes
- Pipetted 2 mL of LB into a 25 mL culture tube with a pre weighed foam sample (negative control)
- Repeat steps 7-10 three more times.
- Set culture tubes with lysate and foam as well as with the control media onto bench top and leave overnight
- Carefully take out foam samples and wash thoroughly with DI water
- Leave washed foam samples to dry in 65º C incubator overnight
- Weigh dried foam samples
