Team:British Columbia/Notebook DspB

Sonication Protocol (with sonicator)

1. Add 100uL of sdH2O to 5mg of lysozyme
2. Pipet this into the cell pellet and resuspend
3. Add 50uL of 0.5M EDTA pH 8.0 to a final concentration of 25uM
4. Add 850uL of TE or EB buffer (fill up to 1mL)
5. Sonicate (ear protectors on!)
1. Turn sonicator on
2. Wipe down probe with kimwipe wetted with ethanol
3. Press start
4. Turn knob up to desired level (in this case, 5)
5. Place mirocentrifuge tube under the probe and move it up and down for 15 seconds
6. Take the tube out and place on ice
7. Repeat 5 more times
8. Turn off sonicator

Sonication Protocol (Lysozyme method)

1. Take the overnight culture out of 37C incubator
2. Transfer the culture into a microcentrifuge tube and spin it down into a pellet
3. Resuspend the culture in approximately 800uL of TE/EB buffer
4. Weigh out lysozyme in microcentrifuge tube (10mg)
5. Dissolve the lysozyme in 100uL of sdH2O
6. Add the 100uL of dissolved lysozyme to the 800uL of culture
7. Place the tube in the 37C incubator for at least 2 hours
8. Centrifuge/spin it down into a pellet
9. Take the supernatant and transfer to another microcentrige tube
10. Use for crude cell assay

Substrate Assay Protocol

1. Take a 96-well plate and assign wells for 6 well replicates for 4 conditions, for a total of 24 wells
2. For the first six wells, add 100uL of phosphate buffer only
3. For the next six wells, add 70uL of phosphate buffer and 30uL of substrate
4. For the next six wells, add 60uL of phosphate buffer, 30uL of substrate, and 10uL of DspB lysate
5. For the final six wells, add 60uL of phosphate buffer, 30uL of substrate, 10uL of control lysate.
6. Take measurements at absorbance wavelength = 405nm using a plate reader. In this case, Tecan M200 Plate Reader is used.

Lessons Learned

1. Make your own aliquots of reagents, especially sdH2O.
2. There's no need to aliquot buffers from kits into a falcon tube before using.
3. You don't need three people to load a gel.
4. You don't need to gel extract your PCR product.

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