Team:TMU-Tokyo/Notebook/Protocol/

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<Br>
<Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment">■Experiment</A></B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment">■Experiment</A></B><Br>
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1st week (8.13 - 8.19)<Br>
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<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/1st_week_(8.13_-_8.19)">1st week (8.13 - 8.19)</A></B><Br>
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2nd week (8.20 - 8.26)<Br>
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<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/2nd_week_(8.20_-_8.26)">2nd week (8.20 - 8.26)</A></B><Br>
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3rd week (8.27 - 9. 2)<Br>
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<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/3rd_week_(8.27_-_9._2)">3rd week (8.27 - 9. 2)</A></B><Br>
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4th week (9. 3 - 9. 9)<Br>
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<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/4th_week_(9._3_-_9._9)">4th week (9. 3 - 9. 9)</A></B><Br>
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5th week (9.10 - 9.16)<Br>
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<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/5th_week_(9.10_-_9.16)">5th week (9.10 - 9.16)</A></B><Br>
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6th week (9.17 - 9.23)<Br>
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<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/6th_week_(9.17_-_9.23)">6th week (9.17 - 9.23)</A></B><Br>
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7th week (9.24 - 9.30)<Br>
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<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/7th_week_(9.24_-_9.30)">7th week (9.24 - 9.30)</A></B><Br>
<Br>
<Br>
<Br>
<Br>
<B>■Protocols</B><Br>
<B>■Protocols</B><Br>
-
Plasmid DNA Purification<Br>
 
-
Genome DNA Purification<Br>
 
-
Restruction Enzyme Degestion<Br>
 
-
DNA Fragment Ligation<Br>
 
-
Transformation<Br>
 
-
Electrophoresis<Br>
 
-
LB Medium<Br>
 
-
<Br>
 
<Br>
<Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay">■Assay</A></B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay">■Assay</A></B><Br>
 +
Assay0<Br>
Device1 Assay<Br>
Device1 Assay<Br>
Device2 Assay<Br>
Device2 Assay<Br>
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<p class="description">
<p class="description">
Our experiment was done follow description.<Br>
Our experiment was done follow description.<Br>
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<Br>
 
<Br>
<Br>
<p class="description">
<p class="description">
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<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>11.</b> Replace the QIAprep Spin Column in a eppendorf tube. Let stand for 1 min.<Br>
<b>11.</b> Replace the QIAprep Spin Column in a eppendorf tube. Let stand for 1 min.<Br>
<b>12.</b> Add 50µL Buffer EB. Let stand for 1 min.<Br>
<b>12.</b> Add 50µL Buffer EB. Let stand for 1 min.<Br>
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<b>12.</b> Centrifuge 1 min. The flow-through is plasmid DNA solution.<Br>
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<b>13.</b> Centrifuge 1 min. The flow-through is plasmid DNA solution.<Br>
<Br>
<Br>
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<Br>
<Br>
<p class="description">
<p class="description">
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<b>■Restruction Enzyme Degestion</b><Br></p>
+
<b>■Restruction Enzyme Digestion</b><Br>
 +
<p class="description3">
 +
<b>1. </b>Mixed following: total 50µL<Br>
 +
(milliQ / DNA solution / Digest buffe /Digest enzyme)<Br>
 +
<b>2. </b>Incubated at 37 °C from 2h to 16h
 +
</p>
<Br>
<Br>
<Br>
<Br>
-
 
<p class="description">
<p class="description">
-
<b>■DNA Fragment Ligation</b><Br></p>
+
<b>■DNA Fragment Ligation</b><Br>
 +
<p class="description3">
 +
<b>1.</b> Mixed following: total 5.0µL
 +
(Vector DNA / Insert DNA / TE )<Br>
 +
<b>2.</b> Added Ligation Mix 5µl.<Br>
 +
<b>3.</b> Incubated at room temperature for 10 minutes.<Br>
<Br>
<Br>
<Br>
<Br>
-
<p class="description">
+
 
-
<b>■Transformation</b><Br></p>
+
</p>
<Br>
<Br>
<Br>
<Br>
<p class="description">
<p class="description">
-
<b>■Electrophoresis</b><Br></p>
+
<b>■Transformation</b><Br>
 +
<p class="description3">
 +
<b>1.</b> Melt competent cells on ice.<Br>
 +
<b>2.</b> Poured DNA solution 10 μl into competent cells calmly.<Br>
 +
<b>3.</b> Incubated on the ice for 40 minutes.<Br>
 +
<b>4.</b> Plated on an agar medium, and then incubated at 37 °C for overnight.<Br>
 +
 
 +
</p>
<Br>
<Br>
<Br>
<Br>
<p class="description">
<p class="description">
-
<b>■LB Medium</b><Br></p>
+
<b>■Electrophoresis</b><Br>
 +
<p class="description3">
 +
<b>1. </b>Set gel in a gel tank. Pour the TAE Buffer into the gel tank.<Br>
 +
<div style="margin-left:20px"><p class="description3">Loading Buffer<Br>
 +
DNA solution<Br>
 +
DW<Br></div>
 +
<p class="description3"><b>2.</b>Mix these components on a Parafilm by pipetting.<Br>
 +
<b>2. </b>Loaded samples into wells.<Br>
 +
<b>3. </b>Start Electrophoresis.<Br>
 +
<b>4. </b>Switch on the power-source and run the gel at 100V for 20min.(stop when samples move to 2/3)<Br>
 +
<b>5. </b>Observe the band by exposing ultraviolet radiation<Br>
 +
 
 +
</p>
<Br>
<Br>
<Br>
<Br>
<p class="description">
<p class="description">
<b>■PCR</b><Br></p>
<b>■PCR</b><Br></p>
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<p class="description">
+
<p class="description3">
-
<Br>・PCR; PrimeSTAR® HS DNA Polymerase<Br>
+
<Br><b>・PCR; PrimeSTAR® HS DNA Polymerase</b><Br>
-
<Br>・Colony PCR ; Takara EX®Taq <Br>
+
<b>1.</b> Add following components on ice.<Br>
-
+
<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)
 +
</div><p class="description3">
 +
<b>2.</b> Dispense PCR solution to PCR tubes.<Br>
 +
<b>3.</b> Set PCR tubes on thermal cycler<Br>
 +
<b>4.</b> Run PCR<Br>
 +
<div style="margin-left:20px"><p class="description3">
 +
・General PCR Conditions <Br>
 +
 
 +
98 ℃・・・5min<Br>
 +
98 ℃・・・10sec.<Br>
 +
55 ℃・・・5 or 15sec.  TM value≧55…5sec. TM value<55…15sec. <Br>
 +
72 ℃・・・1min×1kbp<Br>
 +
72 ℃・・・5min<Br>
 +
4℃・・・∞<Br>
 +
30Cycle
 +
</div>
 +
 
 +
<p class="description3">
 +
 
 +
<Br><b>・Colony PCR ; Takara EX®Taq</b><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)<Br>
 +
DW 15.02 μl<Br>
 +
10× PCR Buffer 2μl <Br>
 +
dNTP (2.5mM) 1.6 μl<Br>
 +
Primer(Forward) 0.64 μl(20µM) <Br>
 +
Primer(Reverse) 0.64 μl(20µM) <Br>
 +
Taq Polymerase 0.1μl  <Br></div>
 +
<p class="description3">
 +
<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>4.</b> Set PCR tubes on thermal cycler <Br>
 +
<b>5.</b> Run PCR <Br></div>
<Br>
<Br>

Latest revision as of 02:29, 27 September 2012

 


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 Digestion

1. Mixed following: total 50µL
(milliQ / DNA solution / Digest buffe /Digest enzyme)
2. Incubated at 37 °C from 2h to 16h



■DNA Fragment Ligation

1. Mixed following: total 5.0µL (Vector DNA / Insert DNA / TE )
2. Added Ligation Mix 5µl.
3. Incubated at room temperature for 10 minutes.




■Transformation

1. Melt competent cells on ice.
2. Poured DNA solution 10 μl into competent cells calmly.
3. Incubated on the ice for 40 minutes.
4. Plated on an agar medium, and then incubated at 37 °C for overnight.



■Electrophoresis

1. Set gel in a gel tank. Pour the TAE Buffer into the gel tank.

Loading Buffer
DNA solution
DW

2.Mix these components on a Parafilm by pipetting.
2. Loaded samples into wells.
3. Start Electrophoresis.
4. Switch on the power-source and run the gel at 100V for 20min.(stop when samples move to 2/3)
5. Observe the band by exposing ultraviolet radiation



■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