Team:HokkaidoU Japan/Protocols

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}
 
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=Protocols=
 
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<ul class="acc" id="acc">
 
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<li>
 
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==Preparation of Competent cells (''E. coli'' DH5a)==
 
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<div class="acc-section">
 
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<div class="acc-content">
 
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===Reagents===
 
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'''TB (Transformation Buffer)(at 4C, filtration)'''
 
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{|border="1" class="protocol"
 
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|
 
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|
 
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|Final concentration
 
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|-
 
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|1 M CaCl<sub>2</sub> (at RT, autoclaved)
 
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|0.75 mL
 
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|15 mM
 
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|-
 
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|4 M KCl (at RT, autoclaved)
 
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|3.125 mL
 
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|250 mM
 
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|-
 
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|1 M MnCl<sub>2</sub> (at 4C, autoclaved)
 
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|2.75 mL
 
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|55 mM
 
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|-
 
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|1 M PIPES (pH 6.7 by NaOH, at 4C, filtration)
 
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|0.5 mL
 
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|10 mM
 
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|-
 
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|'''Total'''
 
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|'''50 mL'''
 
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|
 
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|}
 
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===Procedure===
 
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# Single colony isolation on LB plate
 
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# incubate the plate for 15-19 hrs at 37C
 
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# lift a colony into 2 mL of LB
 
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# culture cells at 37C for 12-16 hrs at 180-200 rpm
 
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# transfer 30 uL, 100 uL, 300 uL of the culture into 100 mL SOB medium, respectively
 
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# culture cells at 20C (for 24 hrs over) at 180-200 rpm (to ΔOD<sub>550nm</sub> = 0.5~0.6)
 
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# leave the 300 mL flask for 10 min on ice
 
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# transfer the culture into two 50 mL Falcon tube
 
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# centrifuge 7500 rpm at 4C for 20 min (TOMY TA-22 rotor), and discard sup
 
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# suspend the pellet in ice-cold 15 mL of TB (Transformation Buffer)(7.5 mL/tube)
 
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# centrifuge 7500 rpm at 4C for 2 min (TOMY TA-22 rotor), and discard sup
 
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# suspend the pellet in ice-cold 3.2 mL of TB
 
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# add 0.24 mL of DMSO (stirring, bit by bit)
 
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# leave the 50 mL Falcon tube for 10 min on ice
 
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# dispense 50 uL into 0.5 mL tube
 
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# freeze the suspension in liquid nitrogen
 
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# store at -80C
 
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</div></div>
 
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</li>
 
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<li>
 
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==Bacterial Transformations==
 
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<div class="acc-section">
 
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<div class="acc-content">
 
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# add DNA solution to thawed competent cells
 
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# incubate the cells on ice for 30 min
 
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# heat shock the cells by immersion in a pre-heated water bath at 42C for 60 sec
 
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# incubate the cells on ice for 5 min
 
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# add 200 uL of SOB broth
 
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# incubate the cells at 37C for 2 hrs while the tubes are shaking
 
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# plate 200 uL of the transformation onto the dish
 
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# incubate the plate at 37C for 12-14 hrs
 
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</div></div>
 
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</li>
 
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<li>
 
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==Mini-prep (Alkaline SDS Method)==
 
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<div class="acc-section"><div class="acc-content">
 
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===Reagents===
 
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'''Solution I'''
 
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(at RT, filtration 0.2 um, 50 mL)
 
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{|border="1" class="protocol"
 
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|-
 
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|
 
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|
 
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|Final concentration
 
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|-
 
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|Glucose (at RT)
 
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|0.45 g
 
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|50 mM
 
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|-
 
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|1 M Tris-HCl (pH8.0, at RT, autoclaved)
 
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|1.25 mL
 
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|25 mM
 
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|-
 
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|0.5 M EDTA (pH8.0, at RT, autoclaved)
 
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|1 mL
 
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|10 mM
 
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|-
 
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|'''Total'''
 
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|'''50 mL'''
 
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|
 
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|}
 
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<br>
 
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'''Solution II'''
 
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(at RT, filtration 0.2 um, 20 mL)
 
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{|border="1" class="protocol"
 
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|-
 
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|
 
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|
 
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|Final concentration
 
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|-
 
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|10 N NaOH (at RT)
 
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|0.4 mL
 
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|0.2 N
 
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|-
 
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|10% SDS (at RT, filtration)
 
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|2 mL
 
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|1%
 
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|-
 
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|'''Total'''
 
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|'''20 mL'''
 
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|
 
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|}
 
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<br>
 
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'''Solution III'''
 
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(at RT, filtration 0.2 um, 50 mL)
 
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{|border="1" class="protocol"
 
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|-
 
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|
 
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|
 
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|Final concentration
 
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|-
 
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|5 M CH<sub>3</sub>COOK
 
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|30 mL
 
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|3 M
 
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|-
 
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|CH<sub>3</sub>COOH
 
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|5.75 mL
 
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|
 
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|-
 
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|H<sub>2</sub>O
 
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|14.25 mL
 
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|
 
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|-
 
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|'''Total'''
 
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|'''50 mL'''
 
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|
 
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|}
 
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===Procedure===
 
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# lift colony ''E. coli'' into 2 mL LB contained antibiotics
 
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# culture cells at 37C for 16-20 hrs at 180-200 rpm
 
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# transfer 1.2-1.5 mL of culture into 1.5 mL tube
 
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# centrifuge the culture at 15,000 rpm for 1 min at 4C and discard sup
 
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# suspend the pellet in ice-cold 100 uL of Solution I
 
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# add 200 uL of Solution II to the suspension
 
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# mix by inverting the tube 10-20 times
 
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# add ice-cold 150 uL of Solution III to the suspension
 
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# mix by inverting the tube 10-20 times
 
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# leave the tube for 5 min on ice
 
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# add 10 uL of Chloroform
 
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# mix by inverting the tube 5-10 times
 
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# centrifuge the suspension at 15,000 rpm for 5 min at 4C
 
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# transfer the supernatant into new 1.5 mL tube↓
 
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# add equal volume of isopropanol and mix by voltexing
 
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# leave the tube for 5 min at RT
 
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# centrifuge the suspension at 15,000 rpm for 10 min at 4C and discard sup
 
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# rinse the ppt by 70% EtOH and mix by voltexing
 
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# centrifuge the suspension at 15,000 rpm for 2 min at 4C and discard sup
 
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# dry up the ppt
 
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# dissolve the ppt in 50 uL of TE (pH 8.0)
 
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# add 1 uL of 10 mg/mL RNase A (4C and stock at –20C)
 
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# incubate for 30 min at 37C
 
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# PCIAA and CIAA extraction
 
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# Ethanol precipitation
 
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# dry up the ppt
 
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# dissolve the ppt in 50 uL of TE (pH 8.0)
 
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</div></div></li><li>
 
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==PCR==
 
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<div class="acc-section"><div class="acc-content">
 
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===Vector===
 
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'''Standard reaction setup'''
 
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{|class="protocol" style="text-align:center;" border="1"
 
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|-
 
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!Component
 
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!Volume
 
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|-
 
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|10x PCR Buffer
 
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|5 uL
 
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|-
 
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|2mM dNTPs
 
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|5 uL
 
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|-
 
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|25mM MgSO<sub>4</sub>
 
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|3 uL
 
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|-
 
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|Suffix-F primer
 
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|1 uL
 
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|-
 
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|Prefix-R primer
 
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|1 uL
 
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|-
 
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|Template DNA
 
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|1 uL
 
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|-
 
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|KOD -Plus- Neo
 
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|1 uL
 
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|-
 
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|DW
 
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|X uL
 
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|-
 
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|'''Total'''
 
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|'''50 uL'''
 
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|}
 
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'''Cycling conditions ''' (2-step cycle)
 
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{|class="protocol" border="1"
 
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|-
 
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| Predenature
 
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| 94C 2 min
 
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|-
 
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| Denature
 
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| 98C 10 sec
 
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|-
 
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| Extension
 
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| 68C X sec (30 sec/kb)
 
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|-
 
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| Hold
 
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| 4C
 
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|}
 
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* 30-40 cycles
 
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===Insert===
 
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'''Standard reaction setup'''
 
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{|class="protocol" style="text-align:center;" border="1"
 
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|-
 
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!Component
 
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!Volume
 
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|-
 
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|10x PCR Buffer
 
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|5 uL
 
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|-
 
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|2mM dNTPs
 
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|5 uL
 
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|-
 
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|25mM MgSO<sub>4</sub>
 
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|3 uL
 
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|-
 
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|EX-F primer
 
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|1 uL
 
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|-
 
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|PS-R primer
 
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|1 uL
 
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|-
 
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|Template DNA
 
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|1 uL
 
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|-
 
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|KOD -Plus- Neo
 
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|1 uL
 
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|-
 
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|DW
 
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|X uL
 
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|-
 
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|'''Total'''
 
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|'''50 uL'''
 
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|}
 
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'''Cycling conditions ''' (2-step cycle)
 
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{|class="protocol" border="1"
 
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|-
 
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| Predenature
 
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| 94C 2 min
 
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|-
 
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| Denature
 
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| 98C 10 sec
 
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|-
 
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| Extension
 
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| 68C X sec (30 sec/kb)
 
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|-
 
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| Hold
 
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| 4C
 
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|}
 
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* 30-40 cycles
 
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===Colony PCR===
 
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* resuspend a colony into 10 uL of DW (template suspension)
 
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*
 
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'''Standard reaction setup'''
 
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{|class="protocol" style="text-align:center;" border="1"
 
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|-
 
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!Component
 
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!Volume
 
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|-
 
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|template suspension
 
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|4.8 uL
 
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|-
 
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|Quick Taq
 
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|5 uL
 
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|-
 
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|Forward primer
 
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|0.1 uL
 
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|-
 
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|Reverse primer
 
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|0.1 uL
 
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|-
 
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|'''Total'''
 
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|'''10 uL'''
 
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|}
 
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'''Cycling conditions ''' (2-step cycle)
 
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{|class="protocol" border="1"
 
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|-
 
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| Predenature
 
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| 94C 2 min
 
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|-
 
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| Denature
 
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| 94C 10 sec
 
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|-
 
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| Extension
 
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| 68C X sec (60 sec/kb)
 
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|-
 
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| Hold
 
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| 4C
 
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|}
 
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* 30-40 cycles
 
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</div></div></li><li>
 
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==Restriction Enzyme Digestions==
 
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<div class="acc-section"><div class="acc-content">c</div></div></li><li>
 
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==DNA ligation==
 
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<div class="acc-section"><div class="acc-content">d</div></div></li><li>
 
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==Agarose gel electrophoresis==
 
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<div class="acc-section"><div class="acc-content">
 
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[[Image:HokkaidoU_Pictures_DNA_Marker.png|200px|thumb|right|DNA Weight Markers]]</div></div></li><li>
 
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==Electroporation==
 
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<div class="acc-section"><div class="acc-content">
 
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'''Preparation of electro-competent cells'''
 
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# cell culture in 400 mL of SOB or LB and grow to ΔOD<sub>600</sub> = 0.5~0.6
 
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# dispense the medium into 8 Falcon 50 mL tube
 
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# centrifuge at 3500 rpm for 5 min at 4C and discard sup
 
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# add 5 mL of DW and suspend the ppt, mix 8 suspensions into single Falcon tube
 
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# centrifuge at 3500 rpm for 5 min at 4C and discard sup
 
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# add 40 mL of DW and suspend the ppt
 
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# centrifuge at 3500 rpm for 5 min at 4C and discard sup
 
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# add 10 mL of 10% Glycerol and suspend the ppt
 
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# centrifuge at 3500 rpm for 5 min at 4C and discard sup
 
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# add 10 mL of 10% Glycerol and suspend the ppt
 
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# centrifuge at 3500 rpm for 5 min at 4C and discard sup
 
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# add 5 mL of 10% Glycerol and suspend the ppt
 
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# dispense 100 uL of the suspensions into 0.5 mL Eppendorf tube, respectively 
 
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# store at -80C freezer
 
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<br>
 
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'''Electroporation'''
 
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</div></div></li><li>
 
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==PCIAA and CIAA extraction==
 
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<div class="acc-section"><div class="acc-content">
 
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'''Reagent'''
 
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* PCIAA = Phenol : Chloroform : IsoAmyl Alcohol = 25 : 24 : 1
 
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* CIAA = Chloroform : IsoAmyl Alcohol = 24 : 1
 
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'''Procedure'''
 
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# add equal volume of PCIAA and vortex vigorously
 
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# centrifuge at 15,000 rpm for 2 min at RT
 
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# transfer the aqueous phase to a new tube, being careful not to transfer the phase interface
 
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# add equal volume of CIAA and vortex vigorously
 
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# transfer the aqueous phase to a new tube
 
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# ethanol precipitation
 
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</div></div></li><li>
 
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==Ethanol presipitation==
 
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<div class="acc-section"><div class="acc-content">
 
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# add 1/10 volume of 3M CH<sub>3</sub>COONa
 
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# add 2.5 volume of 100% ethanol (EtOH)
 
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# incubate on ice for few min
 
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# centrifuge at 15,000 rpm for 10 min at 4C and discard sup
 
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# wash precipitation with 100 uL of 70% EtOH (EtOH has to be cold)
 
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# centrifuge at 15,000 rpm for 5 min at 4C and discard sup
 
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# dry up the ppt (no EtOH should be left)
 
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# resuspend ppt in wanted volume of TE
 
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</div></div></li></ul>
 
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Revision as of 10:17, 25 September 2010

Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Protocols"