Team:TMU-Tokyo/Notebook/Experiment/6th week (9.17 - 9.23)

■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

■6th week (9.17 - 9.23)

■17th Sep
・Densitometry of backbone plasmids
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 no.1: 18 ng/ μl, no.2: 5 ng/ μl.

・Degestion
1. Mixed following: total 50µL (milliQ 3 μl, DNA solution 40 μl, 1 x M buffer 5 μl, XbaI 1 μl, HindIII 1 μl)
2. Incubated at 37 °C for 2 hours.

・Electrophoresis
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 to 2/3.

・Result
We couldn’t obtain the target band.


■18th Sep
Densitometry: backbone(Kanamycin tolerance) plasmids
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 no.1: 12ng/μl, no.2: 80ng/μl

Digestion
1. Mixed following: 50µl
(milliQ 23μl, DNA solution 20μl, 1 x M buffer 5 μl, XbaI 1 μl, HindIII 1 μl)
2. incubated for 2 hours at 37℃.

Electrophoresis
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 to 2/3.

・Result
We can obtain the target band.

・Gel extraction
・Densitometry of backbone(Kanamycin tolerance) plasmids

・Results
Concentration of backbone plasmid no.1: 15ng/μl, fdh4AB: 13ng/μl

・Ligation of fdh4AB
Mixed following: total 6.0 µL (Vector DNA 0.5 μl / Insert DNA 5.5 μl)
・Ligation of Device1
Mixed following: total 5µL
(Vector DNA 0.5 μl / Insert DNA 3.0 μl / TE 1.5 μl)
3. Added Ligation Mix 6 μl to fdh4AB solution and 5μl to Device1
4. Incubated at room temperature for 10 minutes.

・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.
6-1 (fdh4AB) placed on a medium (Amp), incubated at 37℃ for 30 minutes.
6-2 (Device1) added 600 μl LB medium, and then incubated for 30 minutes at 37 °C.
7-2. Plated on an agar medium, and then incubated at 37 °C for overnight.

・Gel-extracted fdh4AB band part PCR (used same DNA as 15th Sep), used new primer for primer F,R
1. 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.1~4 : 10 fold dilution of DNA No.14 ,tube No.5~8 : undiluted DNA No.14, tube No.9~12: 10 fold dilution of DNA No.16, tube No.13~16: undiluted DNA No.16)
2. PCR with Thermal Cycler
(98℃: 5min, 98℃: 10sec, 63℃: 5sec, 72℃: 2min40sec, 72℃: 5min, 4℃: ∞)

・Electrophoresis of PCR product ・Result: Success


■19th Sep
・Densitometry of fdh4AB
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: 358ng/μl, no.2: 40ng/μl, no.3: 35 ng/μl)

・Digestion
1. Mixed following
no.1: total 50µL(milliQ: 35.5µL, DNA solution: 10µL, 1x M Buffer: 2.5µL, XbaⅠ: 1.0µL, HindⅢ: 1.0µL)
no.2: total 50µL(milliQ: 15.5µL, DNA solution: 30µL, 1x M Buffer: 2.5µL, XbaⅠ: 1.0µL, HindⅢ: 1.0µL)
2. incubated for 2 hours at 37℃

・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 of backbone(Kanamycin tolerance) plasmids
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 fdhAB no.1: 55ng/μl, no.2: 23ng/μl

・Ligation of fdh4AB.
1. Mixed following no.1: total 5.0µL (Vector DNA: 0.5µL, Insert DNA: 0.5µL, TE: 3.1µL)
no.2: total 5.0µL (Vector DNA: 0.5µL, Insert DNA: 3.0µL, TE: 1.5µL)

・Ligation of Device1: 1. Mixed following: total 5.0µL
(Vector DNA 0.5 μl, Insert DNA 3.0 μl, TE 1.5 μl)
2. Added Ligation Mix 5 μl to fdh4AB solution and 5μl to Device1
3. Incubated at room temperature for 10 minutes.

・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.
6-1 (fdh4AB) Placed on a medium (Amp), incubated at 37℃ for 30 minutes.
6-2 (Device1) Poured into SOC medium, incubated for 30 minutes at 37℃.
7-2. Plated on an agar medium, and then incubated at 37 °C for overnight.