Team:HokkaidoU Japan/Protocols
From 2010.igem.org
Protocols
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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
- 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
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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
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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
- 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)
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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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Restriction Enzyme Digestions
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DNA ligation
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Agarose gel electrophoresis
File:Image:HokkaidoU Pictures DNA Marker.pngDNA Weight Markers -
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
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Ethanol presipitation
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PCIAA and CIAA extraction
h