Team:HokkaidoU Japan/Protocols
From 2010.igem.org
(Difference between revisions)
(74 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
{{Template:HokkaidoU_Japan}} | {{Template:HokkaidoU_Japan}} | ||
- | + | ||
- | = | + | [[Team:HokkaidoU_Japan/Protocols|English]] |
- | + | / | |
- | + | [[Team:HokkaidoU_Japan/Protocols_jp|日本語]] | |
- | + | ---- | |
- | + | ||
- | + | =HokkaidoU_Japan Protocols= | |
- | + | ||
- | + | [[Team:HokkaidoU_Japan/Protocols#Preparation_of_Competent_cells_.28E._coli_DH5a.29|Preparation of Competent cells (E. coli DH5a)]] | |
- | + | ||
+ | [[Team:HokkaidoU_Japan/Protocols#Bacterial_Transformations|Bacterial Transformations]] | ||
+ | |||
+ | [[Team:HokkaidoU_Japan/Protocols#Mini-prep_.28Alkaline_SDS_Method.29|Mini-prep (Alkaline SDS Method)]] | ||
+ | |||
+ | [[Team:HokkaidoU_Japan/Protocols#PCR|PCR]] | ||
+ | |||
+ | [[Team:HokkaidoU_Japan/Protocols#Electroporation|Electroporation]] | ||
+ | |||
+ | [[Team:HokkaidoU_Japan/Protocols#PCIAA_and_CIAA_extraction|PCIAA and CIAA extraction]] | ||
+ | |||
+ | [[Team:HokkaidoU_Japan/Protocols#Ethanol_presipitation|Ethanol presipitation]] | ||
+ | |||
+ | [[Team:HokkaidoU_Japan/Protocols#Mini-prep_.28QIAprep_Spin_Miniprep_Kit.29|Mini-prep (QIAprep Spin Miniprep Kit)]] | ||
+ | |||
+ | [[Team:HokkaidoU_Japan/Protocols#Gel_Extraction_.28Wizard.C2.AE_SV_Gel_and_PCR_Clean-Up_System.29|Gel Extraction (Wizard® SV Gel and PCR Clean-Up System)]] | ||
+ | |||
+ | [[Team:HokkaidoU_Japan/Protocols#T3SS_RK13_cell_Injection_Assay|T3SS RK13 cell Injection Assay]] | ||
+ | ---- | ||
==Preparation of Competent cells (''E. coli'' DH5a)== | ==Preparation of Competent cells (''E. coli'' DH5a)== | ||
===Reagents=== | ===Reagents=== | ||
- | TB (Transformation Buffer)(at 4C, filtration) | + | '''TB (Transformation Buffer)(at 4C, filtration)''' |
{|border="1" class="protocol" | {|border="1" class="protocol" | ||
| | | | ||
Line 39: | Line 57: | ||
| | | | ||
|} | |} | ||
- | |||
- | === | + | ===Procedure=== |
# Single colony isolation on LB plate | # Single colony isolation on LB plate | ||
- | # | + | # Incubate the plate for 15-19 hrs at 37C |
- | # | + | # Lift a colony into 2 mL of LB |
- | # | + | # Culture cells at 37C for 12-16 hrs at 180-200 rpm |
- | # | + | # Transfer 30 uL, 100 uL, 300 uL of the culture into 100 mL SOB medium, respectively |
- | # | + | # Culture cells at 20C (for 24 hrs over) at 180-200 rpm (to ΔOD<sub>550nm</sub> = 0.5~0.6) |
- | # | + | # Leave the 300 mL flask for 10 min on ice |
- | # | + | # Transfer the culture into two 50 mL Falcon tube |
- | # | + | # Centrifuge 7500 rpm at 4C for 20 min (TOMY TA-22 rotor), and discard sup |
- | # | + | # Suspend the pellet in ice-cold 15 mL of TB (Transformation Buffer)(7.5 mL/tube) |
- | # | + | # Centrifuge 7500 rpm at 4C for 2 min (TOMY TA-22 rotor), and discard sup |
- | # | + | # Suspend the pellet in ice-cold 3.2 mL of TB |
- | # | + | # Add 0.24 mL of DMSO (stirring, bit by bit) |
- | # | + | # Leave the 50 mL Falcon tube for 10 min on ice |
- | # | + | # Dispense 50 uL into 0.5 mL tube |
- | # | + | # Freeze the suspension in liquid nitrogen |
- | # | + | # Store at -80C |
+ | |||
+ | ---- | ||
==Bacterial Transformations== | ==Bacterial Transformations== | ||
+ | # Add DNA solution to thawed competent cells | ||
+ | # Incubate the cells on ice for 30 min | ||
+ | # Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 sec | ||
+ | # Incubate the cells on ice for 5 min | ||
+ | # Add 200 uL of SOB broth | ||
+ | # Incubate the cells at 37C for 2 hrs while the tubes are shaking | ||
+ | # Plate 200 uL of the transformation onto the dish | ||
+ | # Incubate the plate at 37C for 12-14 hrs | ||
+ | |||
+ | |||
+ | ---- | ||
+ | |||
==Mini-prep (Alkaline SDS Method)== | ==Mini-prep (Alkaline SDS Method)== | ||
+ | |||
+ | ===Reagents=== | ||
+ | '''Solution I''' | ||
+ | (at RT, filtration 0.2 um, 50 mL) | ||
+ | {|border="1" class="protocol" | ||
+ | |- | ||
+ | | | ||
+ | | | ||
+ | |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''' | ||
+ | | | ||
+ | |} | ||
+ | <br> | ||
+ | |||
+ | '''Solution II''' | ||
+ | (at RT, filtration 0.2 um, 20 mL) | ||
+ | {|border="1" class="protocol" | ||
+ | |- | ||
+ | | | ||
+ | | | ||
+ | |Final concentration | ||
+ | |- | ||
+ | |10 N NaOH (at RT) | ||
+ | |0.4 mL | ||
+ | |0.2 N | ||
+ | |- | ||
+ | |10% SDS (at RT, filtration) | ||
+ | |2 mL | ||
+ | |1% | ||
+ | |- | ||
+ | |'''Total''' | ||
+ | |'''20 mL''' | ||
+ | | | ||
+ | |} | ||
+ | <br> | ||
+ | |||
+ | '''Solution III''' | ||
+ | (at RT, filtration 0.2 um, 50 mL) | ||
+ | {|border="1" class="protocol" | ||
+ | |- | ||
+ | | | ||
+ | | | ||
+ | |Final concentration | ||
+ | |- | ||
+ | |5 M CH<sub>3</sub>COOK | ||
+ | |30 mL | ||
+ | |3 M | ||
+ | |- | ||
+ | |CH<sub>3</sub>COOH | ||
+ | |5.75 mL | ||
+ | | | ||
+ | |- | ||
+ | |H<sub>2</sub>O | ||
+ | |14.25 mL | ||
+ | | | ||
+ | |- | ||
+ | |'''Total''' | ||
+ | |'''50 mL''' | ||
+ | | | ||
+ | |} | ||
+ | |||
+ | ===Procedure=== | ||
+ | # Lift colony ''E. coli'' into 2 mL LB contained antibiotics | ||
+ | # Culture cells at 37C for 16-20 hrs at 180-200 rpm | ||
+ | # Transfer 1.2-1.5 mL of culture into 1.5 mL tube | ||
+ | # Centrifuge the culture at 15,000 rpm for 1 min at 4C and discard sup | ||
+ | # Suspend the pellet in ice-cold 100 uL of Solution I | ||
+ | # Add 200 uL of Solution II to the suspension | ||
+ | # Mix by inverting the tube 10-20 times | ||
+ | # Add ice-cold 150 uL of Solution III to the suspension | ||
+ | # Mix by inverting the tube 10-20 times | ||
+ | # Leave the tube for 5 min on ice | ||
+ | # Add 10 uL of Chloroform | ||
+ | # Mix by inverting the tube 5-10 times | ||
+ | # Centrifuge the suspension at 15,000 rpm for 5 min at 4C | ||
+ | # Transfer the supernatant into new 1.5 mL tube↓ | ||
+ | # Add equal volume of isopropanol and mix by voltexing | ||
+ | # Leave the tube for 5 min at RT | ||
+ | # Centrifuge the suspension at 15,000 rpm for 10 min at 4C and discard sup | ||
+ | # Rinse the ppt by 70% EtOH and mix by voltexing | ||
+ | # Centrifuge the suspension at 15,000 rpm for 2 min at 4C and discard sup | ||
+ | # Dry up the ppt | ||
+ | # Dissolve the ppt in 50 uL of TE (pH 8.0) | ||
+ | # Add 1 uL of 10 mg/mL RNase A (4C and stock at –20C) | ||
+ | # Incubate for 30 min at 37C | ||
+ | # PCIAA and CIAA extraction | ||
+ | # Ethanol precipitation | ||
+ | # Dry up the ppt | ||
+ | # Dissolve the ppt in 50 uL of TE (pH 8.0) | ||
+ | |||
+ | |||
+ | |||
+ | ---- | ||
+ | |||
==PCR== | ==PCR== | ||
- | == | + | |
- | ==DNA | + | ===Vector=== |
- | == | + | '''Standard reaction setup''' |
+ | {|class="protocol" style="text-align:center;" border="1" | ||
+ | |- | ||
+ | !Component | ||
+ | !Volume | ||
+ | |- | ||
+ | |10x PCR Buffer | ||
+ | |5 uL | ||
+ | |- | ||
+ | |2mM dNTPs | ||
+ | |5 uL | ||
+ | |- | ||
+ | |25mM MgSO<sub>4</sub> | ||
+ | |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) | ||
+ | {|class="protocol" border="1" | ||
+ | |- | ||
+ | | Predenature | ||
+ | | 94C 2 min | ||
+ | |- | ||
+ | | Denature | ||
+ | | 98C 10 sec | ||
+ | |- | ||
+ | | Extension | ||
+ | | 68C X sec (30 sec/kb) | ||
+ | |- | ||
+ | | Hold | ||
+ | | 4C | ||
+ | |} | ||
+ | * 30-40 cycles | ||
+ | ===Insert=== | ||
+ | '''Standard reaction setup''' | ||
+ | {|class="protocol" style="text-align:center;" border="1" | ||
+ | |- | ||
+ | !Component | ||
+ | !Volume | ||
+ | |- | ||
+ | |10x PCR Buffer | ||
+ | |5 uL | ||
+ | |- | ||
+ | |2mM dNTPs | ||
+ | |5 uL | ||
+ | |- | ||
+ | |25mM MgSO<sub>4</sub> | ||
+ | |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) | ||
+ | {|class="protocol" border="1" | ||
+ | |- | ||
+ | | 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''' | ||
+ | {|class="protocol" style="text-align:center;" border="1" | ||
+ | |- | ||
+ | !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) | ||
+ | {|class="protocol" border="1" | ||
+ | |- | ||
+ | | Predenature | ||
+ | | 94C 2 min | ||
+ | |- | ||
+ | | Denature | ||
+ | | 94C 10 sec | ||
+ | |- | ||
+ | | Extension | ||
+ | | 68C X sec (60 sec/kb) | ||
+ | |- | ||
+ | | Hold | ||
+ | | 4C | ||
+ | |} | ||
+ | * 30-40 cycles | ||
+ | |||
+ | |||
+ | ---- | ||
==Electroporation== | ==Electroporation== | ||
+ | |||
+ | ===Preparation of electro-competent cells=== | ||
+ | # Cell culture in 400 mL of SOB or LB and grow to ΔOD<sub>600</sub> = 0.5~0.6 | ||
+ | # Dispense the medium into 8 Falcon 50 mL tube | ||
+ | # Centrifuge at 3500 rpm for 5 min at 4C and discard sup | ||
+ | # Add 5 mL of DW and suspend the ppt, mix 8 suspensions into single Falcon tube | ||
+ | # Centrifuge at 3500 rpm for 5 min at 4C and discard sup | ||
+ | # Add 40 mL of DW and suspend the ppt | ||
+ | # Centrifuge at 3500 rpm for 5 min at 4C and discard sup | ||
+ | # Add 10 mL of 10% Glycerol and suspend the ppt | ||
+ | # Centrifuge at 3500 rpm for 5 min at 4C and discard sup | ||
+ | # Add 10 mL of 10% Glycerol and suspend the ppt | ||
+ | # Centrifuge at 3500 rpm for 5 min at 4C and discard sup | ||
+ | # Add 5 mL of 10% Glycerol and suspend the ppt | ||
+ | # Dispense 100 uL of the suspensions into 0.5 mL Eppendorf tube, respectively | ||
+ | # Store at -80C freezer | ||
+ | |||
+ | ===Electroporation=== | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | ---- | ||
+ | |||
+ | ==PCIAA and CIAA extraction== | ||
+ | |||
+ | ===Reagent=== | ||
+ | * PCIAA = Phenol : CIAA = 1 : 1 | ||
+ | * CIAA = Chloroform : IsoAmyl Alcohol = 24 : 1 | ||
+ | |||
+ | ===Procedure=== | ||
+ | # Add equal volume of PCIAA and vortex vigorously | ||
+ | # Centrifuge at 15,000 rpm for 2 min at RT | ||
+ | # Transfer the aqueous phase to a new tube, being careful not to transfer the phase interface | ||
+ | # Add equal volume of CIAA and vortex vigorously | ||
+ | # Transfer the aqueous phase to a new tube | ||
+ | # Ethanol precipitation | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | ---- | ||
+ | |||
+ | ==Ethanol presipitation== | ||
+ | |||
+ | # Add 1/10 volume of 3M CH<sub>3</sub>COONa | ||
+ | # Add 2.5 volume of 100% ethanol (EtOH) | ||
+ | # Incubate on ice for few min | ||
+ | # Centrifuge at 15,000 rpm for 10 min at 4C and discard sup | ||
+ | # Wash precipitation with 100 uL of 70% EtOH (EtOH has to be cold) | ||
+ | # Centrifuge at 15,000 rpm for 5 min at 4C and discard sup | ||
+ | # Dry up the ppt (no EtOH should be left) | ||
+ | # Resuspend ppt in wanted volume of TE | ||
+ | |||
+ | |||
+ | ---- | ||
+ | |||
+ | ==Mini-prep (QIAprep Spin Miniprep Kit)== | ||
+ | |||
+ | # Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-centrifuge tube | ||
+ | # Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times | ||
+ | # Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times | ||
+ | # Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge | ||
+ | # Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting | ||
+ | # Centrifuge for 30–60 s. Discard the flow-through | ||
+ | # Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through | ||
+ | # Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s | ||
+ | # Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer | ||
+ | # 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)] | ||
+ | |||
+ | |||
+ | ---- | ||
+ | |||
+ | ==Gel Extraction (Wizard® SV Gel and PCR Clean-Up System)== | ||
+ | |||
+ | # Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 mL microcentrifuge tube | ||
+ | # Add 10 uL Membrane Binding Solution per 10 mg of gel slice | ||
+ | # Vortex and incubate at 50–65C until gel slice is completely dissolved | ||
+ | # Insert SV Minicolumn into Collection Tube | ||
+ | # Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly | ||
+ | # Incubate at room temperature for 1 min | ||
+ | # Centrifuge at 16,000 g for 1 min | ||
+ | # Discard flowthrough and reinsert Minicolumn into Collection Tube | ||
+ | # Add 700 uL Membrane Wash Solution (ethanol added) | ||
+ | # Centrifuge at 16,000 g for 1 min | ||
+ | # Discard flowthrough and reinsert Minicolumn into Collection Tube | ||
+ | # Repeat Step 4 with 500 uL Membrane Wash Solution | ||
+ | # Centrifuge at 16,000 g for 5 min | ||
+ | # 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 | ||
+ | # Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube | ||
+ | # Add 50 uL of Nuclease-Free Water to the Minicolumn | ||
+ | # Incubate at room temperature for 1 min | ||
+ | # Centrifuge at 16,000 g for 1 min | ||
+ | # 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)] | ||
+ | |||
+ | |||
+ | ---- | ||
+ | |||
+ | ==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 2x10<sup>5</sup>/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. | ||
+ | # 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 | ||
+ | # 4 hrs later(5.5 hrs before injection) centrifuge the culture 3,500 rpm for 10 min | ||
+ | # 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 | ||
+ | # 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) | ||
+ | # Add 0.4% arabinose 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%CO<sub>2</sub> 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 |
Latest revision as of 08:45, 21 April 2011
English / 日本語
HokkaidoU_Japan Protocols
Preparation of Competent cells (E. coli DH5a)
Mini-prep (Alkaline SDS Method)
Mini-prep (QIAprep Spin Miniprep Kit)
Gel Extraction (Wizard® SV Gel and PCR Clean-Up System)
T3SS RK13 cell Injection Assay
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
- Single colony isolation on LB plate
- Incubate the plate for 15-19 hrs at 37C
- Lift a colony into 2 mL of LB
- Culture cells at 37C for 12-16 hrs at 180-200 rpm
- Transfer 30 uL, 100 uL, 300 uL of the culture into 100 mL SOB medium, respectively
- Culture cells at 20C (for 24 hrs over) at 180-200 rpm (to ΔOD550nm = 0.5~0.6)
- Leave the 300 mL flask for 10 min on ice
- Transfer the culture into two 50 mL Falcon tube
- Centrifuge 7500 rpm at 4C for 20 min (TOMY TA-22 rotor), and discard sup
- Suspend the pellet in ice-cold 15 mL of TB (Transformation Buffer)(7.5 mL/tube)
- Centrifuge 7500 rpm at 4C for 2 min (TOMY TA-22 rotor), and discard sup
- Suspend the pellet in ice-cold 3.2 mL of TB
- Add 0.24 mL of DMSO (stirring, bit by bit)
- Leave the 50 mL Falcon tube for 10 min on ice
- Dispense 50 uL into 0.5 mL tube
- Freeze the suspension in liquid nitrogen
- Store at -80C
Bacterial Transformations
- Add DNA solution to thawed competent cells
- Incubate the cells on ice for 30 min
- Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 sec
- Incubate the cells on ice for 5 min
- Add 200 uL of SOB broth
- Incubate the cells at 37C for 2 hrs while the tubes are shaking
- Plate 200 uL of the transformation onto the dish
- Incubate the plate at 37C for 12-14 hrs
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
- Lift colony E. coli into 2 mL LB contained antibiotics
- Culture cells at 37C for 16-20 hrs at 180-200 rpm
- Transfer 1.2-1.5 mL of culture into 1.5 mL tube
- Centrifuge the culture at 15,000 rpm for 1 min at 4C and discard sup
- Suspend the pellet in ice-cold 100 uL of Solution I
- Add 200 uL of Solution II to the suspension
- Mix by inverting the tube 10-20 times
- Add ice-cold 150 uL of Solution III to the suspension
- Mix by inverting the tube 10-20 times
- Leave the tube for 5 min on ice
- Add 10 uL of Chloroform
- Mix by inverting the tube 5-10 times
- Centrifuge the suspension at 15,000 rpm for 5 min at 4C
- Transfer the supernatant into new 1.5 mL tube↓
- Add equal volume of isopropanol and mix by voltexing
- Leave the tube for 5 min at RT
- Centrifuge the suspension at 15,000 rpm for 10 min at 4C and discard sup
- Rinse the ppt by 70% EtOH and mix by voltexing
- Centrifuge the suspension at 15,000 rpm for 2 min at 4C and discard sup
- Dry up the ppt
- Dissolve the ppt in 50 uL of TE (pH 8.0)
- Add 1 uL of 10 mg/mL RNase A (4C and stock at –20C)
- Incubate for 30 min at 37C
- PCIAA and CIAA extraction
- Ethanol precipitation
- Dry up the ppt
- Dissolve the ppt in 50 uL of TE (pH 8.0)
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
Electroporation
Preparation of electro-competent cells
- Cell culture in 400 mL of SOB or LB and grow to ΔOD600 = 0.5~0.6
- Dispense the medium into 8 Falcon 50 mL tube
- Centrifuge at 3500 rpm for 5 min at 4C and discard sup
- Add 5 mL of DW and suspend the ppt, mix 8 suspensions into single Falcon tube
- Centrifuge at 3500 rpm for 5 min at 4C and discard sup
- Add 40 mL of DW and suspend the ppt
- Centrifuge at 3500 rpm for 5 min at 4C and discard sup
- Add 10 mL of 10% Glycerol and suspend the ppt
- Centrifuge at 3500 rpm for 5 min at 4C and discard sup
- Add 10 mL of 10% Glycerol and suspend the ppt
- Centrifuge at 3500 rpm for 5 min at 4C and discard sup
- Add 5 mL of 10% Glycerol and suspend the ppt
- Dispense 100 uL of the suspensions into 0.5 mL Eppendorf tube, respectively
- Store at -80C freezer
Electroporation
PCIAA and CIAA extraction
Reagent
- PCIAA = Phenol : CIAA = 1 : 1
- CIAA = Chloroform : IsoAmyl Alcohol = 24 : 1
Procedure
- Add equal volume of PCIAA and vortex vigorously
- Centrifuge at 15,000 rpm for 2 min at RT
- Transfer the aqueous phase to a new tube, being careful not to transfer the phase interface
- Add equal volume of CIAA and vortex vigorously
- Transfer the aqueous phase to a new tube
- Ethanol precipitation
Ethanol presipitation
- Add 1/10 volume of 3M CH3COONa
- Add 2.5 volume of 100% ethanol (EtOH)
- Incubate on ice for few min
- Centrifuge at 15,000 rpm for 10 min at 4C and discard sup
- Wash precipitation with 100 uL of 70% EtOH (EtOH has to be cold)
- Centrifuge at 15,000 rpm for 5 min at 4C and discard sup
- Dry up the ppt (no EtOH should be left)
- Resuspend ppt in wanted volume of TE
Mini-prep (QIAprep Spin Miniprep Kit)
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-centrifuge tube
- Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
- Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge
- Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting
- Centrifuge for 30–60 s. Discard the flow-through
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s
- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer
- 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)]
Gel Extraction (Wizard® SV Gel and PCR Clean-Up System)
- Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 mL microcentrifuge tube
- Add 10 uL Membrane Binding Solution per 10 mg of gel slice
- Vortex and incubate at 50–65C until gel slice is completely dissolved
- Insert SV Minicolumn into Collection Tube
- Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly
- Incubate at room temperature for 1 min
- Centrifuge at 16,000 g for 1 min
- Discard flowthrough and reinsert Minicolumn into Collection Tube
- Add 700 uL Membrane Wash Solution (ethanol added)
- Centrifuge at 16,000 g for 1 min
- Discard flowthrough and reinsert Minicolumn into Collection Tube
- Repeat Step 4 with 500 uL Membrane Wash Solution
- Centrifuge at 16,000 g for 5 min
- 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
- Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube
- Add 50 uL of Nuclease-Free Water to the Minicolumn
- Incubate at room temperature for 1 min
- Centrifuge at 16,000 g for 1 min
- 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)]
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.
- 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
- 4 hrs later(5.5 hrs before injection) centrifuge the culture 3,500 rpm for 10 min
- 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
- 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)
- Add 0.4% arabinose 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