InterLab Notebook Documentation, 7/16-8/3

Planned on doing the transformation of the 8 sample plasmids into NEB DH 5-alpha competent E. coli, but couldn't find the cells
Instead, pipetted the 8 sample plasmids out of iGEM 2018 Distribution Kit Plate 7 and suspended in 10 uL of npH2O, then stored in -20 C freezer as follows:

Met with Dr. Velasquez to learn about using the He Lab's plate reader on the 4th floor
Transformed the 8 sample plasmids into NEB 5-alpha competent E. coli according to NEB high-efficiency transformation protocol
Plated original transformation as well as 1:10 dilution for each sample plasmid
Plated on LB + chloramphenicol plates
Incubated at 37 C overnight

Completed three calibration protocols and entered data into the Interlab MichiganState Excel sheet
Prepared two overnight cultures of each of the 8 devices (16 cultures total) as follows:
- 5 mL LB
- 5 uL chloramphenicol
- 1 colony from a plate
- Incubated cultures at 37 C overnight

Performed Day 3 protocol for InterLab:
Measured OD600of 1:10 dilutions of the 16 overnight cultures on the spectrophotometer (blanked with LB + chloramphenicol)
Diluted further to get each culture to target OD600of 0.02
Kept 500 mL of 0 hr cultures in Eppendorf tubes on ice
Put 0.02 OD600cultures back in 37 C incubator for 6 hours
At 6 hours, remove cultures from incubator and take 500 mL samples into Eppendorf tubes on ice
Measured OD600and fluorescence of 4 replicates of each culture at each time point
Data seemed to be off; will re-try later this week

Made overnight cultures for Day 3 re-run tomorrow
- 5 mL LB + chloramphenicol
- 1 colony from plate

Performed Day 3 protocol for InterLab:
Measured OD600of 1:10 dilutions of the 16 overnight cultures on the spectrophotometer (blanked with LB + chloramphenicol)
Diluted further to get each culture to target OD600of 0.02
Kept 500 mL of 0 hr cultures in Eppendorf tubes on ice
Put 0.02 OD600cultures back in 37 C incubator for 6 hours
At 6 hours, remove cultures from incubator and take 500 mL samples into Eppendorf tubes on ice
Measured OD600and fluorescence of 4 replicates of each culture at each time point
Turned in all data to iGEM HQ before the midnight deadline

Made four 3 mL overnight cultures to prepare for CFU protocol tomorrow
- 2 cultures of the negative control device
- 2 cultures of the positive control device
Incubated in 37 C shaker overnight

Performed InterLab CFU Protocol:
Diluted overnight cultures in triplicate to OD600of 0.1 in LB + chloramphenicol media (1 mL total volume)
Performed serial dilutions for each of the 12 cultures and plated dilutions 3, 4, and 5 on LB + chloramphenicol plates
Incubated at 37 C overnight (or so we thought)

Checked plates and saw no sign of growth
- The plates were accidentally incubated at 30 C instead of 37 C, which is why they didn't grow
Incubated 4 more overnight cultures and prepared supplies for tomorrow's repeat of the CFU protocol
New LB+chloramphenicol agar plates were made from heated LB agar + antibiotic
- Plates from yesterday were LB plates with CAM spread on top

Performed InterLab CFU Protocol
Diluted overnight cultures in triplicate to OD600of 0.1 in LB + chloramphenicol media (1 mL total volume)
Performed serial dilutions for each of the 12 cultures and plated dilutions 3, 4, and 5 on LB + chloramphenicol plates
Incubated plates at 37 C overnight

Observed growth on all 36 plates after 18-20 hrs and photographed for evidence and analysis
Colonies were counted on each plate, and results were submitted to and accepted by iGEM HQ
Thus concludes Michigan State iGEM 2018's InterLab experience! 🎉🎉
Seen below: second (A1-Y) and third (A1-Z) replicates of the negative control
- 8x10^6 dilutions in the left column
- 8x10^5 dilutions in the center column
- 8x10^4 dilutions in the right column

Team:MichiganState/InterLab