Team:Technion HS Israel/Protocols

• Home
  Medal Fulfillment Home
• Project More about our project
  Description Results Parts
• Documents
  Experiments Notebook safety Protocols
• Modelling We take our math seriously
  Model Overview Plasmid Loss Equations Parameters Results Documents
• Software
  Software Overview Search Engine Constants Database Simulation About the Database
• Team We are a bunch of nerdy nerds
  Members Attributions and contributions Gallery
• Practices
  Collaborations Practices Search Engine Modeling Tutorials

Protocols

NanoBio Protocol for gene knockout

Antibiotics preparation

Antibiotics preparation

Chemical transformation

DNA Kit Plate

Gel and PCR Extraction

Gel Prep

Glycerol stock

LB SOB SOC BA

Ligation

manual NEB (gibson assembly)

MixandGo

PCR

Phosphorylation and blunt ligation

Presto Mini Plasmid Kit Protocol

Restriction enzyme

Transformation to 3300 LG using electroporation

AHL inducer experiment protocol

• 1. Purpose:
  
  test the response of our bio brick to AHL exposure measuring its fluorescence according to a range of AHL concentrations and determining whether AiiA functions properly.
• 2. Materials:
  • - AHL
  • - Bacteria samples:
    • 2x (+)AiiA
    • 2x (-)AiiA
  • - 2x Eppendorf
  • - LB
  • - Chloramphenicol
• 3. Methods:
  
  Prepare 5 ml starter by growing the cells in 5ml LB medium + appropriate antibiotics (5 µl of chloramphenicol, CM, ) at 37°C overnight.
  
  Samples:
  • (1) +AiiA (K176003)
  • (2) – AiiA (K176005)
  The next day:
  
  
  • 1- Prepare BA+5% (v/v) LB
    • a. 47.5 ml of BA plus 2.5 ml of LB
    • b. Add appropriate antibiotics (chloramphenicol: 50 µl)
  • 2- dilute bacteria 1:100
  
  
  Add 20 µl of bacteria into 2 ml of (BA+5% LB) in a 48 well plate
  
  
  • 3- Add AHL(3OC8) in the 48 well plate in proper concentrations.
  • 4- Incubate it for 2 hours in a shaker ( 37°C, 250 RPM). After 2 hours, measure the absorbance (OD600 – for cell concentration) and fluorescence (excitation peak: 584nm, emission peak: 608nm) using a plate reader. A measurement should be taken every 30 minutes for 3.5 hours (to a total of 7 times).
  
  
  48 well plate AHL(3OC8) concentration preparation:
  
  

  	1	2	3	4	5	6	7	8
  mL	2.2	2	2	2	2	2	2	2
  AHL(3OC8) nM	10000	100	100	10	1	0.1	0.01	0
  AHL(3OC8) µM	10	1	0.1	0.010	0.001	0.0001	0.00001	0
  STEPS
  1	add 0.55 µL
  2	Take 0.2ml	Add here
  3		Take 0.2ml	Add here
  4			Take 0.2ml	Add here
  5				Take 0.2ml	Add here
  6					Take 0.2ml	Add here
  7						Take 0.2ml	Add here
  8							Take 0.2ml
  9	Add 0.19995ml (bacteria with BA and lb)and 0.5µl AHL(3OC8)