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Team:TMU-Tokyo/Notebook/Protocol/
From 2012.igem.org
■Experiment
1st week (8.13 - 8.19)
2nd week (8.20 - 8.26)
3rd week (8.27 - 9. 2)
4th week (9. 3 - 9. 9)
5th week (9.10 - 9.16)
6th week (9.17 - 9.23)
7th week (9.24 - 9.30)
■Protocols
■Assay
Device1 Assay
Device2 Assay
Device3 Assay
Protocol
Our experiment was done follow description.
■Plasmid DNA Purification;
QIAprep Spin Miniprep Kit (QIAGEN)
All centrifuge is done at 13,000rpm, room temperature.
1. Add 1mL overnight cultures of E. coli in LB medium to a eppendorf tube. Centrifuge 1 min and create pellet.
2. Add 250µL Buffer P1 and vortex until pellet perfectly dissolved.
3. Add 250µL Buffer P2 and mix softly by inverting the tube 4-6 times. (Don't vortex.)
4. Add 350µL Buffer N3 and immediately mix softly by inverting the tube 4-6 times. (Don't vortex.)
5. Centrifuge 10 min. Add supernatant to QIAprep Spin Column by pipetting.
6. Centrifuge 1 min. Discard the flow-through.
7. Add 500µL Buffer PB and centrifuge 1 min. Discard the flow-through.
8. Add 400µL Buffer PE and centrifuge 1 min. Discard the flow-through.
9. Repeat step8.
10. Centrifuge 1 min additionally.
11. Replace the QIAprep Spin Column in a eppendorf tube. Let stand for 1 min.
12. Add 50µL Buffer EB. Let stand for 1 min.
13. Centrifuge 1 min. The flow-through is plasmid DNA solution.
■Genome DNA Purification; Generation™ Capture Column (QIAGEN)
1. Add 200µL overnight cultures of E. coli in LB medium to the Capture Column.
2. Incubate 1 min at room temperature.
3. Add 400µL DNA Purification Solution 1, incubate 1 min at room temperature.
4. Centrifuge 10 s with tabletop centrifuge.
5. Transfer the Capture Column to the Waste Collection Tube.
6. Add 400µL DNA Purification Solution 1, incubate 1 min at room temperature.
7. Centrifuge 10 s with tabletop centrifuge.
8. Add 200µl DNA Elution Solution 2. (Don't incubate.)
9. Centrifuge 10 s with tabletop centrifuge.
10. Transfer the Capture Column to DNA Collection Tube.
11. Add 100µl DNA Elution Solution 2, incuvate 10 min at 99℃ with heat block.
12. Centrifuge 20 s with tabletop centrifuge. Purified DNA will be released to solution.
■Restruction Enzyme Degestion
■DNA Fragment Ligation
■Transformation
■Electrophoresis
Put an agalose gel into the tank, and poured TBE buffer.
Mixed DNA samples: loading buffer = 9: 1. Loaded samples into wells.
Electrophoresed, stopped when samples move to 2/3.
■PCR
・PCR; PrimeSTAR® HS DNA Polymerase
1. Add following components on ice.
・General reaction mixture for PCR: 1tube Total 50µL
(5X PrimeSTAR Buffer 10µL / dNTP Mixture 4µL / Primer(Forward) 0.75µL(20µM) / Primer(Reverse) 0.75µL(20µM) / Template DNA(E. coli Genomic DNA 100 pg - 100 ng plasmidDNA 10 pg - 1 ng) / PrimeSTAR HS DNA Polymerase (2.5 U/µL) 0.5µL / Sterilized distilled water up to 50µL)
2. Dispense PCR solution to PCR tubes.
3. Set PCR tubes on thermal cycler
4. Run PCR
・General PCR Conditions
98 ℃・・・5min
98 ℃・・・10sec.
55 ℃・・・5 or 15sec. TM value≧55…5sec. TM value＜55…15sec.
72 ℃・・・1min×1kbp
72 ℃・・・5min
4℃・・・∞
30Cycle
・Colony PCR ; Takara EX®Taq
1. Add the all following components on ice.
・General reaction mixture for Colony PCR (1tube Total 20 μl)
DW 15.02 μl
10× PCR Buffer 2μl
dNTP (2.5mM) 1.6 μl
Primer(Forward) 0.64 μl(20µM)
Primer(Reverse) 0.64 μl(20µM)
Taq Polymerase 0.1μl
2. Dispense PCR solution to PCR tubes.
3. Pick up a colony and put it into PCR tube.then inoculate to master plate.
4. Set PCR tubes on thermal cycler
5. Run PCR
