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Team:TMU-Tokyo/Notebook/Protocol/
From 2012.igem.org
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<p class="description"> | <p class="description"> | ||
| - | <b>■Plasmid DNA Purification</b>; <A Href="http://www.qiagen.com/products/genomicdnastabilizationpurification/generationcapturecolumnkit.aspx"></A> (QIAGEN)<Br></p> | + | <b>■Plasmid DNA Purification</b>; <A Href="http://www.qiagen.com/products/genomicdnastabilizationpurification/generationcapturecolumnkit.aspx"> |
| + | QIAprep Spin Miniprep Kit</A> (QIAGEN)<Br></p> | ||
</p> | </p> | ||
<p class="description3"> | <p class="description3"> | ||
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<b>1.</b> Add following components on ice.<Br> | <b>1.</b> Add following components on ice.<Br> | ||
<div style="margin-left:20px"><p class="description3"> | <div style="margin-left:20px"><p class="description3"> | ||
| - | + | ・General reaction mixture for PCR: 1tube Total 50µL<Br> | |
(5X PrimeSTAR Buffer 10µL / dNTP Mixture 4µL / Primer(Forward) 0.75µL(20µM) / Primer(Reverse) 0.75µL(20µM) / Template DNA(<i>E. coli</i> 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) | (5X PrimeSTAR Buffer 10µL / dNTP Mixture 4µL / Primer(Forward) 0.75µL(20µM) / Primer(Reverse) 0.75µL(20µM) / Template DNA(<i>E. coli</i> 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) | ||
</div><p class="description3"> | </div><p class="description3"> | ||
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<b>4.</b> Run PCR<Br> | <b>4.</b> Run PCR<Br> | ||
<div style="margin-left:20px"><p class="description3"> | <div style="margin-left:20px"><p class="description3"> | ||
| - | + | ・General PCR Conditions <Br> | |
98 ℃・・・5min<Br> | 98 ℃・・・5min<Br> | ||
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<Br><b>・Colony PCR ; Takara EX®Taq</b><Br> | <Br><b>・Colony PCR ; Takara EX®Taq</b><Br> | ||
<b>1.</b> Add the all following components on ice. <Br> | <b>1.</b> Add the all following components on ice. <Br> | ||
| - | + | <div style="margin-left:20px"><p class="description3">・General reaction mixture for Colony PCR (1tube Total 20 μl) | |
DW <Br> | DW <Br> | ||
10× PCR Buffer <Br> | 10× PCR Buffer <Br> | ||
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Primer(Forward) (20µM) <Br> | Primer(Forward) (20µM) <Br> | ||
Primer(Reverse) (20µM) <Br> | Primer(Reverse) (20µM) <Br> | ||
| - | Taq Polymerase <Br> | + | Taq Polymerase <Br></div> |
| + | <p class="description3"> | ||
<b>2.</b> Dispense PCR solution to PCR tubes. <Br> | <b>2.</b> Dispense PCR solution to PCR tubes. <Br> | ||
<b>3.</b> Pick up a colony and put it into PCR tube.then inoculate to master plate. <Br> | <b>3.</b> Pick up a colony and put it into PCR tube.then inoculate to master plate. <Br> | ||
<b>4.</b> Set PCR tubes on thermal cycler <Br> | <b>4.</b> Set PCR tubes on thermal cycler <Br> | ||
| - | <b>5.</b> Run PCR <Br> | + | <b>5.</b> Run PCR <Br></div> |
<Br> | <Br> | ||
Revision as of 19:29, 25 September 2012
■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
Plasmid DNA Purification
Genome DNA Purification
Restruction Enzyme Degestion
DNA Fragment Ligation
Transformation
Electrophoresis
LB Medium
■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
■LB Medium
■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
10× PCR Buffer
dNTP (2.5mM)
Primer(Forward) (20µM)
Primer(Reverse) (20µM)
Taq Polymerase
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
