"

From 2010.igem.org

Revision as of 06:34, 27 October 2010 (view source) Sprkata (Talk | contribs) ← Older edit		Revision as of 06:35, 27 October 2010 (view source) Sprkata (Talk | contribs) Newer edit →
Line 215:		Line 215:
	|-		|-
	|}		|}
-
-	==Methods==
-
-	===Type III secretion system RK13 cell Injection Assay===
-	'''Seed RK13 cells'''
-	# Remove the culture medium and wash 3 times PBS followed by trypsinization
-	# Suspend RK13 cells with antibiotics free RPMI-10% FCS
-	# Seed 6 x 10<sup>5</sup>/well RK13 on the 6 well plate coated with poly-L-Lysin 24 hrs before infection
-
-	'''Prepare ''E. coli'' culture'''
-	# Grow ''E. coli'' K12 (SPI2/Signal-GFP), ''E. coli'' K12 (SPI2) and ''E. coli'' K12 (Signal-GFP) in 4 mL of LB (+0.4% Arabinose) with appropriate antibiotics at 37C overnight
-
-	'''5 hrs before injection'''
-	# Centrifuge 4 mL of ''E. coli'' culture at 3,500 rpm for 10 min in the round tube
-	# Discard the supernatant and resuspend with 4 mL of MgM-MES(pH 5.0) + 0.4% Arabinose with appropriate antibiotics and grow at 37C for 4 hrs
-	# 4 hrs later centrifuge the culture 3,500 rpm for 10 min and discard the sup
-	# Resuspend the pellet with 2 mL of antibiotics free RPMI-10% FCS and transfer the culture into micro tube
-	# Centrifuge the culture 6,000 rpm for 3 min and discard the sup
-	# Repeat step 4 and 5 three times
-	# Measure and adjust the concentration of E. coli RPMI culture (ΔOD = 0.5)
-
-	'''Injection'''
-	# Remove the RPMI on RK13
-	# Add 900 uL of fresh RPMI-10% FCS and then 100 uL of E. coli RPMI culture (ΔOD = 0.5)
-	# Incubate the plate at 37C for 1 hrs, 5% CO<sub>2</sub> with or without shaking
-	# Wash the wells with penisilin/ strepromicin containing RPMI-10% FCS twice
-	# Add 2 mL of penisilin/ streptomicin containing RPMI-10% FCS/well
-	# Incubate the plate at 37C for 1 hr, 5% CO<sub>2</sub>
-	# Observe the cells by fluorescence microscope under blue light at every 30 min

---

Revision as of 06:35, 27 October 2010