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

STEP 3: 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 l into a cuvette and adjusted 0.
4.Threw the solvent, poured the DNA sample.
5.Measured the concentration.


Results

Concentration of fdh4AB was 20ng/µl.

Digestion
1.Mixed following
fdh4AB
milliQ 　　　　　　　　5.5µl
DNA solution 40µl
10×K buffer 2.5µl
BamHⅠ 1.0µl
SacⅠ 　1.0µl
Total 50µl

2.Incubated for 2 hours at 37°C.

Gel extraction

Densitometry
Concentration of fdh4AB was 2ng/µl.

Ligation
1.Mixed following .
Vector DNA 4µl
Insert DNA 0.5µl
TE 0.5µl
Total 5.0µl

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

STEP 1: 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. 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 l into a cuvette and adjusted 0.
4.Threw the solvent, poured the DNA sample.
5.Measured the concentration.

Results
Concentration of fdh4AB was 20ng/µl.

Digestion
1.Mixed following
fdh4AB
milliQ 　　　　　　　　5.5µl
DNA solution 40µl
10×K buffer 2.5µl
BamHⅠ 1.0µl
SacⅠ 　1.0µl
Total 50µl
2.Incubated for 2 hours at 37°C.
Gel extraction

Densitometry
Concentration of fdh4AB was 2ng/µl.

Ligation
1.Mixed following .
Vector DNA 4µl
Insert DNA 0.5µl
TE 0.5µl
Total 5.0µl
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


STEP 1: Construction of Device3
Colony PCR of 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


STEP 1: Construction of Device3
PCR of insert (fdh4AB)
Electrophoresis
Success: We observed appropriate bands but a lot of smear
Colony PCR of 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
STEP 1: 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 was 8 ng/ μl. no.2 was 13 ng/ μl. Digestion 1. Mixed following milliQ 7.5 μl DNA solution 38 μl 0.5 x K buffer 2.5 μl SacI 1 μl BamHI 1 μl Total 50 μl 2. Incubated at 37 °C for 2 hours. 3. Heat inactivated for 20 minutes at 50 °C. 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 was 18 ng/ μl. no.2 was 5 ng/ μl. 1. Mixed following milliQ 3 μl DNA solution 40 μl 1 x M buffer 5 μl XbaI 1 μl HindIII 1 μl Total 50 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.