Team:TMU-Tokyo/Notebook/Experiment/4th week (9. 3 - 9. 9)

(Construction of Device1)
・Colony PCR of transformed E. coli (pfrm-frmR-GFP)
・Electrophoresis
→Failure: because of no bands


・Digestion
1. Mixed following
fdh4AB: total 50µL
(milliQ 5.5µL / DNA solution 40µL / 10×K buffer 2.5µL / BamH 1.0µL / SacⅠ 1.0µL
2.Incubated for 2 hours at 37°C.

・Electrophoresis
1.Put an agarose gel into the tank, and poured TBE buffer.
2.Mixed DNA sample : loading buffer = 9 : 1. Loaded sample into wells.
3.Electrophoresed, stopped when sample move 2/3.

・Results
There was no target band.

・Electrophoresis of PCR products: before digestion of fdh4AB
→diluted 10 times and 50 times of PCR products of fdh4AB.
1.Put an agarose gel into the tank, and poured TBE buffer.
2.Mixed DNA sample : loading buffer = 9 : 1. Loaded samples into wells.
3.Electrophoresed, stopped when samples move 2/3.
・Results
There was the target band.

・Gel extraction
1. Excised the DNA fragment from an agarose gel. For each 100mg of agarose gel added 300µl Buffer NT.
2. Incubated sample for 5-10 minutes at 50°C. Vortexed the sample briefly every 2-3 minutes until the gel slice was completely dissolved.
3. Placed a NucleoSpin Extract ⅡColumn into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000×g. Discarded flow-though and placed the column back into the collection tube.
4. Added 700 µL Buffer NT3 to the NucleoSpin Extact II Column. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
5. Centrifuged for 2 minutes at 11,000 x g to remove Buffer NT3 completely.
6. Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 µL Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.

・Densitometry
1.Diluted DNA samples 50 times with a solvent.
2.Turned on the machine; GeneQuant 100.
3.Poured the solvent 100 µL into a cuvette and adjusted 0.
4.Threw the solvent, poured the DNA sample.
5.Measured the concentration.
・Results
Concentration of fdh4AB was 40ng/µl.

・Ligation
1. Mixed following: total 5µL
(Vector DNA 1µL / Insert DNA 1.6µL / TE 2.4µL) 2. Added Ligation Mix 5µl.
3. Incubated at room temperature for 10 minutes.

・Transformation
1. Melt competent cells on ice.
2. Poured DNA solution 10µl into competent cells calmly.
3. Incubated on the ice for 40 minutes.
4. Heat shocked at 42°C for 45 seconds.
5. Incubated on the ice for 2-3 minutes.
6. Plated on an agar medium, and then incubated at 37°C for overnight.

・Result There was no colony.

■ 5th Sep