Team:TMU-Tokyo/Notebook/Protocol/

■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 50 μl)
5X PrimeSTAR Buffer 10 μl
dNTP Mixture 4 μl
Primer F 0.75μl（20μM）
Primer R 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) 0.5 μl
Sterilized distilled water up to 50 μl

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