"
Team:HokkaidoU Japan/Protocols/Infection Assay
From 2011.igem.org
(Difference between revisions)
m |
m |
||
| Line 14: | Line 14: | ||
===10 hrs before injection=== | ===10 hrs before injection=== | ||
# Centrifuge 4 mL of ''E. coli'' culture at 3,500 rpm for 10 min in the round tube. | # Centrifuge 4 mL of ''E. coli'' culture at 3,500 rpm for 10 min in the round tube. | ||
| - | # | + | # Centrifuge the culture 3,500 rpm for 10 min |
| - | + | # Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0) with appropriate antibiotics and grow for 4 hrs at 37C | |
| - | # Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0) | + | |
# 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup | # 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup | ||
# Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube | # Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube | ||
| Line 22: | Line 21: | ||
# Repeat step 6 and 7 three times | # Repeat step 6 and 7 three times | ||
# Measure and adjust the concentration of ''E. coli'' RPMI(pH 5.0) culture(ΔOD = 0.06) | # Measure and adjust the concentration of ''E. coli'' RPMI(pH 5.0) culture(ΔOD = 0.06) | ||
| - | # | + | # And appropriate antibiotics into this ''E. coli'' culture |
===Injection=== | ===Injection=== | ||
Revision as of 03:36, 6 October 2011
HokkaidoU Japan
iGEM 2011 Team of Hokkaido University
Contents |
T3SS RK13 cell Injection Assay
Seed RK13 cells
- Remove the culture medium and wash 3 times with PBS follwed by trypsinization
- Suspend RK13 cells with antibiotics free RPMI-10% FCS
- Seed 2x105/well RK13 cells on a 6 well plate or a glass bottom 3.5 cm dish 20 hrs before infection
Prepare E. coli culture
- Grow E.coli-K12(SPI2/Signal-GFP/RFP), and E. coli K12(SPI2)in 4 mL of LB+0.4% arabinose with appropriate antibiotics at 37C overnight
10 hrs before injection
- Centrifuge 4 mL of E. coli culture at 3,500 rpm for 10 min in the round tube.
- Centrifuge the culture 3,500 rpm for 10 min
- Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0) with appropriate antibiotics and grow for 4 hrs at 37C
- 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup
- Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube
- Centrifuge the culture 5,000 rpm for 2 min and discard the sup
- Repeat step 6 and 7 three times
- Measure and adjust the concentration of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
- And appropriate antibiotics into this E. coli culture
Injection
- Remove the RPMI on RK13
- Add 1 mL of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
- Incubate the plate at 37C, 5%CO2 and observe the cells by Cofocal Laser Scannning Microscope(OLYMPUS FV-1000D) under blue and green exciter light at every 1.5 hrs after first exposure.
