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

STEP 1: Construction of Device3
PCR of insert (fdh4AB)
Electrophoresis
Failure: because of too dilute bands
PCR of insert (fdh4AB)
Electrophoresis
Failure: because of annealing temperature
PCR of insert (fdh4AB)
Electrophoresis
Success: We observed appropriate bands!

Electrophoresis of PCR products: before digestion of fdh4AB, after PCR products

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

Results
One sample appeared a band.

Gel extraction
1.Excised the DNA fragment from an aglose gel. For each 100mg of agalose gel added 200µl 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 30 seconds at 11,000×g. Discarded flow-through and placed the column back into the collection tube.
4.Added 700µl Buffer NT3 to the NucleoSpin Extract ⅡColumn. Centrifuged for 30 seconds at 11,000×g . Discarded flow-through and placed the column back into the collection tube.
5.Centrifuged for 1 minute at 11,000×g.

Densitometry
1.Diluted DNA sample 50 times with a solvent.
2.Turned on the machine: GeneQuant 100
3.Poured the solvent 100µl into a cuvette and adjusted 0.
4.Threw the solvent, poured the DNA sample.
5.Measured the concentration.

Results
Concentration of fdh4AB was 38ng/µl


Ligation 1.Mixed following .
① ②
Vector DNA 1µl 1µl
Insert DNA 1.6µl 2.3µl
TE 2.4µl 1.7µl
Total 5µl 5µl

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°Cfor 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.

Results
There was some colony.

■ 4th Sep