Team:TMU-Tokyo/Notebook/Experiment/2nd week (8.20 - 8.26)

■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)

Experiment

■2nd week (8.20 - 8.26)

■21th August
(Construction of Device1)
・Purification Escherichia coli genome DNA Densitometry of purified
・PCR of insert (pfrm-frmR)
・Preparation of 0.8 % Agarose Gel
・Electrophoresis
→Failure: because of too dilute bands
・PCR of insert (pfrm-frmR)
・Preparation of 0.8 % Agarose Gel

■22nd Aug
(Construction of Device1)
・Electrophoresis
→Failure: because of no bands
・PCR of insert (pfrm-frmR)
・Preparation of 0.8 % Agarose Gel
・Electrophoresis
→Failure: because of not appropriate bands
・PCR of vector (BBa_K299009)
・Preparation of 0.8 % Agarose Gel
・Electrophoresis
→Success: We observed appropriate bands!

・Refine of PCR products of frmR
Mixed 1 volume of sample with 2 volumes of Buffer NT.
Placed a NucleoSpin Extract II Column into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Added 700 μl Buffer NT3 to the NucleoSpin Extract II Column. Centrifuged for 30 seconds at 11,000 x g. Discarded flow-though and placed the column back into the collection tube.
Centrifuged for 1 minute at 11,000 x g to remove Buffer NT3 completely.
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 11,000x g.

・Digestion
1. Mixed following: total 50µL
(milliQ 17.5µL / DNA solution 28µL / 0.5x K buffe 2.5µL / BamHI 1µL / SacI 1µL)
2. Incubated for 1 hour at 37 °C.


■23rd Aug
・Refine of frmR and backbone plasmid
Mixed 1 volume of sample with 2 volumes of Buffer NT.
Placed a NucleoSpin Extract II Column into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Added 700 μl Buffer NT3 to the NucleoSpin Extract II Column. Centrifuged for 30 seconds at 11,000 x g. Discarded flow-though and placed the column back into the collection tube.
Centrifuged for 1 minute at 11,000 x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 40 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.

・Densitometry
Diluted DNA samples 50 times with a solvent.
Turned on the machine; GeneQuant 100.
Poured the solvent 100 μl into a cuvette and adjusted 0.
Threw the solvent, poured the DNA sample.
Measured the concentration.

・Results
Concentration of frmR: 35 ng/ µL.
Backbone plasmid: 105 ng/ µL.

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

・Results
We couldn’t obtain the target band of frmR.


■24th Aug
(Construction of Device1)
・PCR of insert (pfrm-frmR)
・Preparation of 0.8 % Agarose Gel
・Electrophoresis
→Success: We observed appropriate bands but a lot of smere.
・Gradient PCR of insert (pfrm-frmR)


・Densitometry of fdh4AB PCR product.
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 fhd4AB no.1: 123 ng/ µL, no.2: 110 ng/ µL/

・Digestion
1. Mixed following: total 50µL
(milliQ 25.5µL / DNA solution 20µL / 0.5x K buffe 2.5µL / BamHI 1µL / SacI 1µL)
2. Incubated at 37 °C for 2.5 hours.

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

・Results
We couldn’t obtain the target bands.
We run electrophoresis for the samples of before digestion, but we couldn’t obtain the target bands.


■25th Aug
・Electrophoresis of gradient PCR products of fdh4AB
Put an agalose gel into the tank, and poured TBE buffer.
Mixed DNA samples: loading buffer = 9: 1. Loaded samples into wells.
Electrophoresed, stopped when samples move to 2/3.

Samples
68 C
67.1 C
65.5 C
63.1 C
60.1 C
58.0 C
56.2 C
55.0 C

・Results
We obtained the target bands from F, G and H. Also E showed a weak band. These data showed that better annealing temperature was 55.0 – 58.0 C.