"
Team:TMU-Tokyo/Notebook/Protocol/
From 2012.igem.org
| Line 131: | Line 131: | ||
<p class="description"> | <p class="description"> | ||
<b>■PCR</b><Br></p> | <b>■PCR</b><Br></p> | ||
| - | <p class=" | + | <p class="description3"> |
| - | <Br>・PCR; PrimeSTAR® HS DNA Polymerase< | + | <Br><b>・PCR; PrimeSTAR® HS DNA Polymerase</b><Br> |
| - | <Br> | + | 1. Add following components on ice.<Br> |
| - | 1. 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) | |
| - | 5X PrimeSTAR Buffer | + | </div><p class="description3"> |
| - | dNTP Mixture | + | 2. Dispense PCR solution to PCR tubes.<Br> |
| - | Primer(Forward) 0. | + | 3. Set PCR tubes on thermal cycler<Br> |
| - | + | 4. Run PCR<Br> | |
| - | + | <div style="margin-left:20px"><p class="description3"> | |
| - | PrimeSTAR HS DNA Polymerase (2.5 U/ | + | |
| - | Sterilized distilled water up to | + | |
| - | < | + | |
| - | 2. Dispense PCR solution to PCR tubes.<Br> | + | |
| - | 3. Set PCR tubes on thermal cycler<Br> | + | |
| - | 4. Run PCR<Br> | + | |
General PCR Conditions <Br> | General PCR Conditions <Br> | ||
| Line 157: | Line 151: | ||
4℃・・・∞<Br> | 4℃・・・∞<Br> | ||
30Cycle | 30Cycle | ||
| + | </div> | ||
| + | <p class="description3"> | ||
| - | + | <Br><b>・Colony PCR ; Takara EX®Taq</b><Br> | |
| - | + | 1. Add the all following components on ice. | |
| - | <Br>・Colony PCR ; Takara EX®Taq <Br> | + | |
| - | 1. Add the all following components on ice. | + | |
General reaction mixture for Colony PCR (1tube Total 20 μl) | General reaction mixture for Colony PCR (1tube Total 20 μl) | ||
DW | DW | ||
| Line 170: | Line 164: | ||
& Reverse)0.64 (20µM) | & Reverse)0.64 (20µM) | ||
Taq Polymerase 0.01 | Taq Polymerase 0.01 | ||
| - | 2. Dispense PCR solution to PCR tubes. | + | 2. Dispense PCR solution to PCR tubes. |
| - | 3. Pick up a colony and put it into PCR tube.then inoculate to master plate. | + | 3. Pick up a colony and put it into PCR tube.then inoculate to master plate. |
| - | 4. Set PCR tubes on thermal cycler | + | 4. Set PCR tubes on thermal cycler |
| - | 5. Run PCR | + | 5. Run PCR |
5 | 5 | ||
Revision as of 17:34, 24 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; (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 Buffer2
dNTP (2.5mM)1.6
Primer(Forward 0.64
& Reverse)0.64 (20µM)
Taq Polymerase 0.01
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
5
4
1.6
1.6
0.25
