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HokkaidoU_Japan Protocols

Preparation of Competent cells (E. coli DH5a)

Bacterial Transformations

Mini-prep (Alkaline SDS Method)

PCR

Electroporation

PCIAA and CIAA extraction

Ethanol presipitation

Mini-prep (QIAprep Spin Miniprep Kit)

Gel Extraction (Wizard® SV Gel and PCR Clean-Up System)

T3SS RK13 cell Injection Assay

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Preparation of Competent cells (E. coli DH5a)

Reagents

TB (Transformation Buffer)(at 4C, filtration)

		Final concentration
1 M CaCl2 (at RT, autoclaved)	0.75 mL	15 mM
4 M KCl (at RT, autoclaved)	3.125 mL	250 mM
1 M MnCl2 (at 4C, autoclaved)	2.75 mL	55 mM
1 M PIPES (pH 6.7 by NaOH, at 4C, filtration)	0.5 mL	10 mM
Total	50 mL

Procedure

1. Single colony isolation on LB plate
2. Incubate the plate for 15-19 hrs at 37C
3. Lift a colony into 2 mL of LB
4. Culture cells at 37C for 12-16 hrs at 180-200 rpm
5. Transfer 30 uL, 100 uL, 300 uL of the culture into 100 mL SOB medium, respectively
6. Culture cells at 20C (for 24 hrs over) at 180-200 rpm (to ΔOD_550nm = 0.5~0.6)
7. Leave the 300 mL flask for 10 min on ice
8. Transfer the culture into two 50 mL Falcon tube
9. Centrifuge 7500 rpm at 4C for 20 min (TOMY TA-22 rotor), and discard sup
10. Suspend the pellet in ice-cold 15 mL of TB (Transformation Buffer)(7.5 mL/tube)
11. Centrifuge 7500 rpm at 4C for 2 min (TOMY TA-22 rotor), and discard sup
12. Suspend the pellet in ice-cold 3.2 mL of TB
13. Add 0.24 mL of DMSO (stirring, bit by bit)
14. Leave the 50 mL Falcon tube for 10 min on ice
15. Dispense 50 uL into 0.5 mL tube
16. Freeze the suspension in liquid nitrogen
17. Store at -80C

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Bacterial Transformations

1. Add DNA solution to thawed competent cells
2. Incubate the cells on ice for 30 min
3. Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 sec
4. Incubate the cells on ice for 5 min
5. Add 200 uL of SOB broth
6. Incubate the cells at 37C for 2 hrs while the tubes are shaking
7. Plate 200 uL of the transformation onto the dish
8. Incubate the plate at 37C for 12-14 hrs

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Mini-prep (Alkaline SDS Method)

Reagents

Solution I (at RT, filtration 0.2 um, 50 mL)

		Final concentration
Glucose (at RT)	0.45 g	50 mM
1 M Tris-HCl (pH8.0, at RT, autoclaved)	1.25 mL	25 mM
0.5 M EDTA (pH8.0, at RT, autoclaved)	1 mL	10 mM
Total	50 mL

Solution II (at RT, filtration 0.2 um, 20 mL)

		Final concentration
10 N NaOH (at RT)	0.4 mL	0.2 N
10% SDS (at RT, filtration)	2 mL	1%
Total	20 mL

Solution III (at RT, filtration 0.2 um, 50 mL)

		Final concentration
5 M CH3COOK	30 mL	3 M
CH3COOH	5.75 mL
H2O	14.25 mL
Total	50 mL

Procedure

1. Lift colony E. coli into 2 mL LB contained antibiotics
2. Culture cells at 37C for 16-20 hrs at 180-200 rpm
3. Transfer 1.2-1.5 mL of culture into 1.5 mL tube
4. Centrifuge the culture at 15,000 rpm for 1 min at 4C and discard sup
5. Suspend the pellet in ice-cold 100 uL of Solution I
6. Add 200 uL of Solution II to the suspension
7. Mix by inverting the tube 10-20 times
8. Add ice-cold 150 uL of Solution III to the suspension
9. Mix by inverting the tube 10-20 times
10. Leave the tube for 5 min on ice
11. Add 10 uL of Chloroform
12. Mix by inverting the tube 5-10 times
13. Centrifuge the suspension at 15,000 rpm for 5 min at 4C
14. Transfer the supernatant into new 1.5 mL tube↓
15. Add equal volume of isopropanol and mix by voltexing
16. Leave the tube for 5 min at RT
17. Centrifuge the suspension at 15,000 rpm for 10 min at 4C and discard sup
18. Rinse the ppt by 70% EtOH and mix by voltexing
19. Centrifuge the suspension at 15,000 rpm for 2 min at 4C and discard sup
20. Dry up the ppt
21. Dissolve the ppt in 50 uL of TE (pH 8.0)
22. Add 1 uL of 10 mg/mL RNase A (4C and stock at –20C)
23. Incubate for 30 min at 37C
24. PCIAA and CIAA extraction
25. Ethanol precipitation
26. Dry up the ppt
27. Dissolve the ppt in 50 uL of TE (pH 8.0)

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PCR

Vector

Standard reaction setup

Component	Volume
10x PCR Buffer	5 uL
2mM dNTPs	5 uL
25mM MgSO4	3 uL
Suffix-F primer	1 uL
Prefix-R primer	1 uL
Template DNA	1 uL
KOD -Plus- Neo	1 uL
DW	X uL
Total	50 uL

Cycling conditions (2-step cycle)

Predenature	94C 2 min
Denature	98C 10 sec
Extension	68C X sec (30 sec/kb)
Hold	4C

• 30-40 cycles

Insert

Standard reaction setup

Component	Volume
10x PCR Buffer	5 uL
2mM dNTPs	5 uL
25mM MgSO4	3 uL
EX-F primer	1 uL
PS-R primer	1 uL
Template DNA	1 uL
KOD -Plus- Neo	1 uL
DW	X uL
Total	50 uL

Cycling conditions (2-step cycle)

Predenature	94C 2 min
Denature	98C 10 sec
Extension	68C X sec (30 sec/kb)
Hold	4C

• 30-40 cycles

Colony PCR

• resuspend a colony into 10 uL of DW (template suspension)

Standard reaction setup

Component	Volume
template suspension	4.8 uL
Quick Taq	5 uL
Forward primer	0.1 uL
Reverse primer	0.1 uL
Total	10 uL

Cycling conditions (2-step cycle)

Predenature	94C 2 min
Denature	94C 10 sec
Extension	68C X sec (60 sec/kb)
Hold	4C

• 30-40 cycles

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Electroporation

Preparation of electro-competent cells

1. Cell culture in 400 mL of SOB or LB and grow to ΔOD₆₀₀ = 0.5~0.6
2. Dispense the medium into 8 Falcon 50 mL tube
3. Centrifuge at 3500 rpm for 5 min at 4C and discard sup
4. Add 5 mL of DW and suspend the ppt, mix 8 suspensions into single Falcon tube
5. Centrifuge at 3500 rpm for 5 min at 4C and discard sup
6. Add 40 mL of DW and suspend the ppt
7. Centrifuge at 3500 rpm for 5 min at 4C and discard sup
8. Add 10 mL of 10% Glycerol and suspend the ppt
9. Centrifuge at 3500 rpm for 5 min at 4C and discard sup
10. Add 10 mL of 10% Glycerol and suspend the ppt
11. Centrifuge at 3500 rpm for 5 min at 4C and discard sup
12. Add 5 mL of 10% Glycerol and suspend the ppt
13. Dispense 100 uL of the suspensions into 0.5 mL Eppendorf tube, respectively
14. Store at -80C freezer

Electroporation

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PCIAA and CIAA extraction

Reagent

• PCIAA = Phenol : CIAA = 1 : 1
• CIAA = Chloroform : IsoAmyl Alcohol = 24 : 1

Procedure

1. Add equal volume of PCIAA and vortex vigorously
2. Centrifuge at 15,000 rpm for 2 min at RT
3. Transfer the aqueous phase to a new tube, being careful not to transfer the phase interface
4. Add equal volume of CIAA and vortex vigorously
5. Transfer the aqueous phase to a new tube
6. Ethanol precipitation

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Ethanol presipitation

1. Add 1/10 volume of 3M CH₃COONa
2. Add 2.5 volume of 100% ethanol (EtOH)
3. Incubate on ice for few min
4. Centrifuge at 15,000 rpm for 10 min at 4C and discard sup
5. Wash precipitation with 100 uL of 70% EtOH (EtOH has to be cold)
6. Centrifuge at 15,000 rpm for 5 min at 4C and discard sup
7. Dry up the ppt (no EtOH should be left)
8. Resuspend ppt in wanted volume of TE

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Mini-prep (QIAprep Spin Miniprep Kit)

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-centrifuge tube
2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting
6. Centrifuge for 30–60 s. Discard the flow-through
7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min

• [http://www.qiagen.com/products/plasmid/qiaprepminiprepsystem/qiaprepspinminiprepkit.aspx see details (Official website)]

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Gel Extraction (Wizard® SV Gel and PCR Clean-Up System)

1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 mL microcentrifuge tube
2. Add 10 uL Membrane Binding Solution per 10 mg of gel slice
3. Vortex and incubate at 50–65C until gel slice is completely dissolved
4. Insert SV Minicolumn into Collection Tube
5. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly
6. Incubate at room temperature for 1 min
7. Centrifuge at 16,000 g for 1 min
8. Discard flowthrough and reinsert Minicolumn into Collection Tube
9. Add 700 uL Membrane Wash Solution (ethanol added)
10. Centrifuge at 16,000 g for 1 min
11. Discard flowthrough and reinsert Minicolumn into Collection Tube
12. Repeat Step 4 with 500 uL Membrane Wash Solution
13. Centrifuge at 16,000 g for 5 min
14. Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol
15. Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube
16. Add 50 uL of Nuclease-Free Water to the Minicolumn
17. Incubate at room temperature for 1 min
18. Centrifuge at 16,000 g for 1 min
19. Discard Minicolumn and store DNA at 4C or –20C

• [http://www.promega.com/applications/pcr/featuresandbenefits/Wizard_SV_Gel_PCR_Clean-Up_System.htm see details (Official website)]

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T3SS RK13 cell Injection Assay

Seed RK13 cells

1. Remove the culture medium and wash 3 times with PBS follwed by trypsinization
2. Suspend RK13 cells with antibiotics free RPMI-10% FCS
3. 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

1. 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

1. Centrifuge 4 mL of E. coli culture at 3,500 rpm for 10 min in the round tube.
2. Discard the sup and resuspend with 4 mL of MgM-MES(pH 7.2)+0.4% arabinose with appropriate antibiotics and grow for 4 hrs at 37C
3. 4 hrs later(5.5 hrs before injection) centrifuge the culture 3,500 rpm for 10 min
4. Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0)+0.4% arabinose with appropriate antibiotics and grow for 4 hrs at 37C
5. 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup
6. Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube
7. Centrifuge the culture 5,000 rpm for 2 min and discard the sup
8. Repeat step 6 and 7 three times
9. Measure and adjust the concentration of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
10. Add 0.4% arabinose and appropriate antibiotics into this E. coli culture

Injection

1. Remove the RPMI on RK13
2. Add 1 mL of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
3. 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