Team:TMU-Tokyo/Notebook/Experiment/5th week (9.10 - 9.16)

■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

■5th week (9.10 - 9.16)

■10th Sep
(Construction of Device2)
・PCR of insert (FDH)
・Electrophoresis
→Failure: because of a lot of smear
・Ordered Device1 and Device2 plasmids arrive!

Electrophoresis of PCR products: before digestion of fdh4AB.
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
There was the target band.

・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 fdh4AB: 20ng/µl.

・Digestion
1. Mixed following: total 50µL
(milliQ 5.5µl / DNA solution(fdh4AB) 40µ / 10×K buffer 2.5µl / BamHⅠ 1.0µl / SacⅠ 1.0µl
2. Incubated for 2 hours at 37°C.

・Gel extraction
・Densitometry
Concentration of fdh4AB: 2ng/µl.

・Ligation
1. Mixed following: total 5.0µl
(Vector DNA 4µl / Insert DNA 0.5µl / TE 0.5µl
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°C for 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.


■11th Sep

(Construction of Device3)
・PCR of insert (fdh4AB)
・Electrophoresis
→Failure: because of no bands

・Electrophoresis of PCR products: before digestion of fdh4AB.
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.

・Gel extraction of fdh4AB 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.< Br> 4. 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.
5. Centrifuged for 2 minutes at 11,000 x g to remove Buffer NT3 completely.
6. 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
There was the target band.

・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 fdh4AB: 20ng/µl.

・Digestion
1. Mixed following: total 50µL
(milliQ 5.5µl / DNA solution(fdh4AB) 40µl / 10×K buffer 2.5µl / BamHⅠ 1.0µl SacⅠ 1.0µl)
2.Incubated for 2 hours at 37°C.

・Gel extraction
・Densitometry
Concentration of fdh4AB: 2ng/µl.

・Ligation
1. Mixed following: total 5.0µl
(Vector DNA 4µl / Insert DNA 0.5µl / TE 0.5µl)
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°C for 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.


■12th Sep


(Construction of Device3)
・Colony PCR: transformed E. coli (p-fdh4AB)
・Electrophoresis
→Failure: because of transformation miss
・Electrophoresis
→Failure: because of transformation miss
・Electrophoresis
→Failure: because of transformation miss
・Electrophoresis
→Failure: because of transformation miss


■13th Sep

(Construction of Device3)
・PCR of insert (fdh4AB)
・Electrophoresis
→Success: we observed appropriate bands but a lot of smear
・Colony PCR: transformed E. coli (p-fdh4AB)
・Electrophoresis
→Failure: because of no appropriate bands

Transformation
1. Melt competent cells on ice.
2. Poured DNA solution (pSB1K3) 10 µl into competent cells calmly
3. Incubated on the ice for 40 minutes
4. Heat shocked at 42 °C for 45 seconds
5. Incubated on the ice for 2-3 minutes
6. Added 600 µl LB medium, and then incubated for 30 minutes at 37 °C
7. Plated on an agar medium, and then incubated at 37 °C for overnight


■14th Sep
New fdh4AB primers arrive
(Construction of Device3)
・PCR of insert (fdh4AB)
・Electrophoresis
→Failure: because of artificial mistake?
・PCR of insert (fdh4AB)
・Electrophoresis
→Success: appropriate bands are clearer than before!

・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.
4. 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.
5. Centrifuged for 2 minutes at 11,000 x g to remove Buffer NT3 completely.
6. 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 fdh4AB no.1: 8 ng/ µl, no.2: 13 ng/ µl.

・Digestion
1. Mixed following: total 50µl
(milliQ 7.5 µl / DNA solution 38 µl / 0.5x K buffer 2.5 µl / SacⅠ 1 µl / BamHⅠ 1 µl)

2. Incubated at 37 °C for 2 hours.
3. Heat inactivated for 20 minutes at 50 °C.


■15th Sep
・Gel-extracted fdh4AB band part PCR The densitometry result: DNA No.14･･･40μg/μl, DNA No.16…8μg/μl
・ Made PCR solution
tube4 ×4set 50μl ( DW 140.5 / Buffer 42.5 / dNTP 17 /pF 3.1875 / pR 3.1875 /DNA 4 / Poly 2.125)
10 fold dilution of DNA (DW: DNA=18:2)
tube No.25~28 : undiluted DNA No.14 ,tube No.29~32 : 10 fold dilution of DNA No.14, tube No.33~36 : undiluted DNA No.16, tube No.37~40: 10 fold dilution of DNA No.16)
・ PCR with Thermal Cycler: (98℃: 5min, 98℃: 10sec, 63℃: 5sec, 72℃: 2min40sec, 72℃: 5min, 4℃: ∞)
・ Electrophoresis of PCR product
・ Result
PCR product was seen only on undiluted DNA No.14 but it was smearing