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

■Assay
Assay0
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 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.

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.

2. Dispense PCR solution to PCR tubes.
3. Set PCR tubes on thermal cycler
4. Run PCR

・Colony PCR ; Takara EX®Taq
1. Add the all following components on ice.

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