Difference between revisions of "Team:MIT/InterlabStudy"
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5. Interrogate by flow cytometry | 5. Interrogate by flow cytometry | ||
| − | M9 Min w/ Glycerol | + | <nowiki>M9 Min w/ Glycerol |
• 20 mL 5X m9 | • 20 mL 5X m9 | ||
• 3.4 mL 10mg/mL thiamine | • 3.4 mL 10mg/mL thiamine | ||
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• 2 mL 10% cas AA | • 2 mL 10% cas AA | ||
• 0.2 mL 1 M MgSO4 | • 0.2 mL 1 M MgSO4 | ||
| − | • 10 uL 1 M CaCl2 | + | • 10 uL 1 M CaCl2</nowiki> |
Mix, and then add DI water until the total volume is 100 mL (for a total of 100 mL) | Mix, and then add DI water until the total volume is 100 mL (for a total of 100 mL) | ||
Revision as of 04:13, 23 August 2015
Introduction
The iGEM Interlab Study aims “is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world”. It is an opportunity for teams throughout the world to build and characterize parts. The purpose of the study as a whole is to be able to “test the consistency of the teams’ data of the measured devices”.
Devices Built and Measured: (Chassis : E coli)
Device 1 : J23101 + I13504 (B0034-E0040-B0015) -> backbone:PSB1C3 (3 biological replicates)
Device 2 : J23106 + I13504 (B0034-E0040-B0015) -> backbone:PSB1C3 (3 biological replicates)
Device 3 : J23117 + I13504 (B0034-E0040-B0015) -> backbone: PSB1C3 (3 biological replicates)
Measurement Controls:
Positive Control: I20270 PSB1C3 -> J23151 + I20270(B0032-E0040-B0010-B0012)
Negative Control: BBa_R0040
Negative Control : NEB 10 B Competent Cells
Protocols:
DNA Kit Plate Instructions (http://parts.igem.org/Help:2015_DNA_Distribution)
Transformation (http://parts.igem.org/Help:Protocols/Transformation)
Making Liquid cultures: • Prepare culture in a 15 mL, round bottom tube. • Add 5mL LB using a seriological pipette • Add 5uL of 1000x antibiotic (Chloramphenicol.) • Pick colony using a 10 ul pipette tip on a p2. Eject tip into tube (tip should remain in tube). • if growing from another liquid culture, 100 uL should be plenty (replacing the 1 colony). Almost no amount is too small, just ensure that you get cells. Miniprep – using Qiagen protocol (http://www.hawaii.edu/microbiology/MO/docs/diversity/Qia-Miniprep.pdf)
3 Antibiotic Assembly protocol – (http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf))
Transformation (http://parts.igem.org/Help:Protocols/Transformation)
Colony PCR (selecting transformants from the transformation plates) – (https://www.neb.com/~/media/NebUs/Files/Application%20Notes/appNoteM0486.pdf)
Making Liquid Culture (see above)
Generalized E coli Flow Cytometry Protocol( created by Nicholas Delateur Weiss Lab MIT)
1. Grow an overnight in rich media with appropriate antibiotic from a single colony (in previous step)
2. Subculture 125 uL of saturated culture into 5 mL of M9 Minimal Media (below) supplemented with Glycerol, add appropriate antibiotic
3. Grow with shaking at desired temperature until desired OD (0.3 for exponential phase, 1.0ish for steady phase)
4. Dilute 10 uL into 990 uL 1X PBS
5. Interrogate by flow cytometry
M9 Min w/ Glycerol • 20 mL 5X m9 • 3.4 mL 10mg/mL thiamine • 0.8 mL 50% glycerol • 2 mL 10% cas AA • 0.2 mL 1 M MgSO4 • 10 uL 1 M CaCl2 Mix, and then add DI water until the total volume is 100 mL (for a total of 100 mL)
Measuring in flow cytometer
- First we measured with beads
- Then with samples