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)


■Protocols
Plasmid DNA Purification
Genome DNA Purification
Restruction Enzyme Degestion
DNA Fragment Ligation
Transformation
Electrophoresis
LB Medium


■Assay
Device1 Assay
Device2 Assay
Device3 Assay

Experiment

■2nd week (8.20 - 8.26)

■22nd Aug
・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
milliQ 17.5 μl
DNA solution 28 μl
0.5 x K buffer 2.5 μl
BamHI 1 μl
SacI 1 μl Total 50 μl
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 was 35 ng/ μl.
Backbone plasmid was 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
Densitometry of fdh4AB.
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 fhd4AB no.1 was 123 ng/ μl.
no.2 was 110 ng/ μl/

Digestion
1. Mixed following
milliQ 25.5 μl
DNA solution 20 μl
0.5 x K buffer 2.5 μl
BamHI 1 μl
SacI 1 μl Total 50 μl
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.
Results
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.