Team:TMU-Tokyo/Notebook/Experiment/3rd week (8.27 - 9. 2)

■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

■3rd week (8.27 - 9. 2)

■27th Aug
・Refine of PCR products of frmR and fdh4AB
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 1 minute 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 1 minute at 11,000 x g. Discarded flow-though and placed the column back into the collection tube.
Centrifuged for 2 minutes 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 45 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: 85 ng/ µL, fdh4AB: 43 ng/ µL.

・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 1 minute 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 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Centrifuged for 2 minutes 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.

・Densitometry
Concentration of frmR: 53 ng/ µL, fdh4AB: 45 ng/ µL.

・Digestion of frmR and fdh4AB
1. Mixed following: total 50µL
(milliQ 5.5µL / DNA solution 20µL / 0.5x K buffe 2.5µL / SacI 1µL / BamHI 1µL)
2. Incubated at 37 °C for 2 hours.

・Gel extraction

・Densitometry
Concentration of frmR: 45 ng/ µL, fdh4AB: 32 ng/ µL.


■28th Aug
Digestion of backbone plasmid.
1. Mixed following:: total 25µL
(milliQ 3.55 µL / DNA solution 18.2 µL / 0.5x K buffer 1.25 µL / SacI 1 µL / BamHI 1 µL)
2. Incubated at 37 °C for 2 hours.

・Gel extraction
1. Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.
2. Incubated sample for 5 – 10 minutes at 50 °C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.
3. Placed a NucleoSpin Extract II Columu into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
5. 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.
6. Centrifuged for 2 minutes 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.

・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 backbone plasmid 30 ng/ µL.

・Ligation of frmR
1.1 Mixed following: total 5 µL
(Vector DNA 1 µL / Insert DNA 1.6 µL / TE 2.4 µL)
・Ligation of fdh4AB
1.2 Mixed following: total 6 µL
(Vector DNA 1 µL / Insert DNA 2.3 µL / TE 2.7 µL)
2. Added Ligation Mix 5 μl.
3. Incubated at room temperature for 30 minutes.

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

・Results
Colony didn’t appear.


■29th Aug
・Ligation of frmR
1.1 Mixed following: total 5 µL
(Vector DNA 1 µL / Insert DNA 1.6 µL / TE 2.4 µL)
・Ligation of fdh4AB
1.2 Mixed following: total 6 µL
(Vector DNA 1 µL / Insert DNA 2.3 µL / TE 2.7 µL)
2. Added Ligation Mix 5 μl.
3. Incubated at room temperature for 15 minutes.

・Transformation
1. Melt competent cells on ice.
2. Poured DNA solution 10 μl into competent cells calmly.
3. Incubated on the ice for 40 minutes.
4. Heat shocked at 42 °C for 30 seconds.
5. Plated on an agar medium, and then incubated at 37 °C for overnight.

・Results
Colony didn’t appear.


■30th Aug
(Construction of Device1)
・PCR of insert (pfrm-frmR)
・Preparation of 0.8 % Agarose Gel
・Electrophoresis
→Success: We observed appropriate bands!


・Ligation
1.1 frmR: Mixed following: total 5µL
(Vector DNA 1µL / Insert DNA 1.6µl / TE 2.4 µL)
1.2 fdh4AB: Mixed following：total 5 µL (Vector DNA 1 µL / Insert DNA 2.3 µL / TE 1.7 µL)
2. Added Ligation Mix 5 μl.
3. Incubated at room temperature for 10 minutes.

・Transformation
1. Melt competent cells on ice.
2. Poured DNA solution 10 μl into competent cells calmly.
3. Incubated on the ice for 40 minutes.
4. Heat shocked at 42 °C for 30 seconds
5. Incubated on the ice for 2 minutes.
6. Plated on an agar medium, and then incubated at 37 °C for overnight.

・Results
Colony didn’t appear.

■31th Aug
New fdh4AB primers arrive
(Construction of Device3)
・PCR of insert (fdh4AB)
・Electrophoresis
→Failure: We observed appropriate bands but a lot of nonspecific amplification products