Team:TMU-Tokyo/Notebook/Experiment/1st week (8.13 - 8.19)

■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

■1st week (8.13 - 8.19)

■ 14th Aug
(Construction of Device3) ・PCR of vector (BBa_K299009) and insert (fdh4AB) ・Preparation of 1.5 % Agarose Gel Electrophoresis
→Failure: because of Preparation of Gel protocol miss

・Preparation of 1.5 % Agarose Gel Electrophoresis (again)
→Failure: because of Preparation of Gel protocol miss

・Preparation of 1.5 % Agarose Gel
→Failure: because of PCR protocol miss

・PCR of vector (BBa_K299009) and insert (fdh4AB) ・Preparation of 0.8 % Agarose Gel ・Correction of the PCR protocol and Preparation of Gel protocol


■15th Aug

(Construction of Device3)
・Electrophoresis of previous sample
→Failure: because of PCR protocol miss
・PCR of vector (BBa_K299009) and insert (fdh4AB)
・Preparation of 0.8 % Agarose Gel Electrophoresis
→Failure: because of smear
・PCR of vector (BBa_K299009)

・Digestion
1. Mixed following: total 20µL
(DW 15µL / DNA solution 2µL / 10x L buffer 2µL / SacI 1µL;)
2. Incubated at 37 °C for 1 hour.
3. Heat inactivated at 65 °C for 10 minutes.
4. Mixed: Total 50µL
(DW 24µL / Reaction solution 20µL / 10x K buffer 5µL / BamHI 1µL;)
5. Incubated at 37 °C for 1 hour 40 minutes.

・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.

・Gel extraction
Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200µL Buffer NT.
Incubated sample for 5 – 10 minutes at 50 C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.
Placed a NucleoSpin Extract II Columu into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000x g. Discarded flow-through and placed the column backed into the collection tube.
Added 700µL Buffer NT3 to the NucleoSpin Extact II Column. Centrifuged for 30 s at 11,000x g. Discarded flow-through and placed the column backed into the collection tube.
Centrifuged for 1 min at 11,000x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcetrifuge tube. Added 30µL Buffer NE and incubated at room temperature for 1 min. Centrifuged for 1 min at 11,000x g.

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

・Results
Concentration of a back bone plasmid was 53 ng /μl.
We could not obtain the target fragments of fdh4AB.


■16th Aug

(Construction of Device3)
・Preparation of 0.8 % Agarose Gel Electrophoresis
→Success: We observed appropriate bands!
・PCR of insert (fdh4AB) ・Check the fdh4AB primer’s sequence


・Refine of PCR products: fdh4AB 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 s at 11,000x g. Discarded flow-through and placed the column back into the collection tube.
Added 700µL Buffer NT3 to the NucleoSpin Extract II Column. Centrifuged for 30 s at 11,000x g. Discarded flow-though and placed the column back into the collection tube.
Centrifuged for 1 minute at 11,000x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 30µL Buffer NE and incubated at room temperature for 1 min. Centrifuged for 1 minute at 11,000x 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 fdh4AB was 210 ng/ μl.
Backbone plasmid No. 01 was 110 ng/ μl
No. 02 was 60 ng/ μl

・Digestion
1. Mixed following
Fdh4AB: total 25µL (milliQ 6.5µL / DNA solution 15µL / 10x L buffer 2.5µL / SacI 1µL)
Backbone plasmid: total 25µL (milliQ 1.5µL / DNA solution 20µL / 10x L buffer 2.5µL / SacI 1µL)
2. Incubated for 1 hour at 37 °C.
3. Heat inactivated at 65 °C for 10 minutes.
4. Mixed following; total 50µL (milliQ 19µL / Reaction solution 25 µL / 10x K buffer 5µL / BamHI 1µL)
5. Incubated at 37 °C for 1 hour.

・Gel extraction
Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.
Incubated sample for 5 – 10 minutes at 50 °C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.
Placed a NucleoSpin Extract II Columu 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 Extact II Column. Centrifuged for 30 seconds at 11,000 x g. Discarded flow-through 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 11,000 x g.

・Results
We could not obtain the target band of fdh4AB.

・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 backbone plasmid was 48 ng/ μl.


■17th Aug

(Construction of Device3)
・Preparation of 0.8 % Agarose Gel ・Electrophoresis →Failure: because of different bands ・PCR of insert (fdh4AB) ・Preparation of 0.8 % Agarose Ge

Electrophoresis of PCR products: before digestion of fdh4AB, after PCR products clean-up of fdh4AB, before PCR products clean-up of fdh4AB and diluted 100 times of 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.

・Results
Every sample was smeared. It seemed that PCR didn’t run well.

■18th Aug

(Construction of Device3)
・Electrophoresis
→Failure: because of deterioration of Loading buffer
・Preparation of 0.8 % Agarose Gel
・Electrophoresis
→Failure: because of miss of fdh4AB primer’s sequence
・Redesign and Order new fdh4AB primer