Team:TMU-Tokyo/Notebook/Experiment/4th week (9. 3 - 9. 9)
From 2012.igem.org
Line 50: | Line 50: | ||
<b>■3rd September<b/></p> | <b>■3rd September<b/></p> | ||
<p class="description3"> | <p class="description3"> | ||
- | + | (Construction of Device3)<Br> | |
- | + | ・PCR of insert (fdh4AB), electrophoresis<Br> | |
- | + | →Failure: because of too dilute bands<Br> | |
- | + | ・PCR of insert (fdh4AB), electrophoresis<Br> | |
- | + | →Failure: because of annealing temperature<Br> | |
- | + | ・PCR of insert (fdh4AB), electrophoresis<Br> | |
- | + | →Success: We observed appropriate bands!<Br> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<Br> | <Br> | ||
+ | <b>・Electrophoresis of PCR products:</b> before digestion of fdh4AB, after PCR products<Br> | ||
1.Put an agalose gel into the tank, and poured TBE buffer.<Br> | 1.Put an agalose gel into the tank, and poured TBE buffer.<Br> | ||
2.Mixed DNA samples : loading buffer = 9 : 1. Loaded samples into wells.<Br> | 2.Mixed DNA samples : loading buffer = 9 : 1. Loaded samples into wells.<Br> | ||
3.Electrophoresed stopped when samples move 2/3.<Br> | 3.Electrophoresed stopped when samples move 2/3.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Results</b><Br> | |
One sample appeared a band.<Br> | One sample appeared a band.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Gel extraction</b><Br> | |
- | 1.Excised the DNA fragment from an aglose gel. For each 100mg of agalose gel added 200µl Buffer NT.<Br> | + | 1. Excised the DNA fragment from an aglose gel. For each 100mg of agalose gel added 200µl Buffer NT.<Br> |
- | 2.Incubated sample for 5-10 minutes at 50°C. Vortexed the sample briefly every 2-3 minutes until the gel slice was completely dissolved.<Br> | + | 2. Incubated sample for 5-10 minutes at 50°C. Vortexed the sample briefly every 2-3 minutes until the gel slice was completely dissolved.<Br> |
- | 3.Placed a NucleoSpin Extract ⅡColumn into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000×g. Discarded flow-through and placed the column back into the collection tube.<Br> | + | 3. Placed a NucleoSpin Extract ⅡColumn into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000×g. Discarded flow-through and placed the column back into the collection tube.<Br> |
- | 4.Added 700µl Buffer NT3 to the NucleoSpin Extract ⅡColumn. Centrifuged for 30 seconds at 11,000×g . Discarded flow-through and placed the column back into the collection tube.<Br> | + | 4. Added 700µl Buffer NT3 to the NucleoSpin Extract ⅡColumn. Centrifuged for 30 seconds at 11,000×g . Discarded flow-through and placed the column back into the collection tube.<Br> |
- | 5.Centrifuged for 1 minute at 11,000×g. <Br> | + | 5. Centrifuged for 1 minute at 11,000×g. <Br> |
<Br> | <Br> | ||
- | + | <b>・Densitometry</b><Br> | |
- | 1.Diluted DNA sample 50 times with a solvent.<Br> | + | 1. Diluted DNA sample 50 times with a solvent.<Br> |
- | 2.Turned on the machine: GeneQuant 100<Br> | + | 2. Turned on the machine: GeneQuant 100<Br> |
- | 3.Poured the solvent 100µl into a cuvette and adjusted 0.<Br> | + | 3. Poured the solvent 100µl into a cuvette and adjusted 0.<Br> |
- | 4.Threw the solvent, poured the DNA sample.<Br> | + | 4. Threw the solvent, poured the DNA sample.<Br> |
- | 5.Measured the concentration.<Br> | + | 5. Measured the concentration.<Br> |
<Br> | <Br> | ||
- | + | <b>・Results</b><Br> | |
Concentration of fdh4AB was 38ng/µl<Br> | Concentration of fdh4AB was 38ng/µl<Br> | ||
<Br> | <Br> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
<Br> | <Br> | ||
- | 2.Added Ligation Mix | + | <b>・Ligation</b><Br> |
- | 3.Incubated at room temperature for 10 minutes.<Br> | + | 1. Mixed following .<Br> |
+ | No.1: Total 5µL (Vector DNA 1µL / Insert DNA 1.6µL / TE 2.4µL)<Br> | ||
+ | No.2: Total 5µL (Vector DNA 1µL / Insert DNA 2.3µL / TE 1.7µL)<Br> | ||
+ | 2. Added Ligation Mix 5µL.<Br> | ||
+ | 3. Incubated at room temperature for 10 minutes.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Transformation</b><Br> | |
- | 1.Melt competent cells on ice.<Br> | + | 1. Melt competent cells on ice.<Br> |
- | 2.Poured DNA solution 10µl into competent cells calmly.<Br> | + | 2. Poured DNA solution 10µl into competent cells calmly.<Br> |
- | 3.Incubated on the ice for 40 minutes.<Br> | + | 3. Incubated on the ice for 40 minutes.<Br> |
- | 4.Heat shocked at 42°Cfor 45 seconds.<Br> | + | 4. Heat shocked at 42°Cfor 45 seconds.<Br> |
- | 5.Incubated on the ice for 2-3 minutes.<Br> | + | 5. Incubated on the ice for 2-3 minutes.<Br> |
- | 6.Plated on an agar medium, and then incubated at 37°C for overnight.<Br> | + | 6. Plated on an agar medium, and then incubated at 37°C for overnight.<Br> |
<Br> | <Br> | ||
- | + | <b>・Results</b><Br> | |
There was some colony.<Br> | There was some colony.<Br> | ||
<Br> | <Br> | ||
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<b>■ 4th Sep<b/></p> | <b>■ 4th Sep<b/></p> | ||
<p class="description3"> | <p class="description3"> | ||
- | + | (Construction of Device1)<Br> | |
- | + | ・Colony PCR of transformed E. coli (pfrm-frmR-GFP)<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of no bands<Br> | |
<Br> | <Br> | ||
<Br> | <Br> | ||
- | + | <b>・Digestion</b><Br> | |
- | 1.Mixed following<Br> | + | 1. Mixed following<Br> |
- | fdh4AB<Br> | + | fdh4AB: total 50µL<Br> |
- | milliQ | + | (milliQ 5.5µL / DNA solution 40µL / 10×K buffer 2.5µL / BamH 1.0µL / SacⅠ 1.0µL<Br> |
- | DNA solution | + | |
- | 10×K buffer | + | |
- | + | ||
- | SacⅠ | + | |
- | + | ||
- | <Br> | + | |
2.Incubated for 2 hours at 37°C.<Br> | 2.Incubated for 2 hours at 37°C.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Electrophoresis</b><Br> | |
1.Put an agarose gel into the tank, and poured TBE buffer.<Br> | 1.Put an agarose gel into the tank, and poured TBE buffer.<Br> | ||
2.Mixed DNA sample : loading buffer = 9 : 1. Loaded sample into wells.<Br> | 2.Mixed DNA sample : loading buffer = 9 : 1. Loaded sample into wells.<Br> | ||
3.Electrophoresed, stopped when sample move 2/3.<Br> | 3.Electrophoresed, stopped when sample move 2/3.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Results</b><Br> | |
There was no target band.<Br> | There was no target band.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Electrophoresis of PCR products:</b> before digestion of fdh4AB<Br> | |
→diluted 10 times and 50 times of PCR products of fdh4AB.<Br> | →diluted 10 times and 50 times of PCR products of fdh4AB.<Br> | ||
- | |||
1.Put an agarose gel into the tank, and poured TBE buffer.<Br> | 1.Put an agarose gel into the tank, and poured TBE buffer.<Br> | ||
2.Mixed DNA sample : loading buffer = 9 : 1. Loaded samples into wells.<Br> | 2.Mixed DNA sample : loading buffer = 9 : 1. Loaded samples into wells.<Br> | ||
3.Electrophoresed, stopped when samples move 2/3.<Br> | 3.Electrophoresed, stopped when samples move 2/3.<Br> | ||
- | + | <b>・Results</b><Br> | |
There was the target band.<Br> | There was the target band.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Gel extraction</b><Br> | |
- | 1.Excised the DNA fragment from an agarose gel. For each 100mg of agarose gel added 300µl Buffer NT.<Br> | + | 1. Excised the DNA fragment from an agarose gel. For each 100mg of agarose gel added 300µl Buffer NT.<Br> |
- | 2.Incubated sample for 5-10 minutes at 50°C. Vortexed the sample briefly every 2-3 minutes until the gel slice was completely dissolved.<Br> | + | 2. Incubated sample for 5-10 minutes at 50°C. Vortexed the sample briefly every 2-3 minutes until the gel slice was completely dissolved.<Br> |
- | 3.Placed a NucleoSpin Extract ⅡColumn into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000×g. Discarded flow-though and placed the column back into the collection tube.<Br> | + | 3. Placed a NucleoSpin Extract ⅡColumn into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000×g. Discarded flow-though and placed the column back into the collection tube.<Br> |
- | 4.Added 700 | + | 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.<Br> |
- | 5.Centrifuged for 2 minutes at 11,000 x g to remove Buffer NT3 completely.<Br> | + | 5. Centrifuged for 2 minutes at 11,000 x g to remove Buffer NT3 completely.<Br> |
- | 6.Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 | + | 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.<Br> |
- | + | ||
- | <Br> | + | |
<Br> | <Br> | ||
+ | <b>・Densitometry</b><Br> | ||
1.Diluted DNA samples 50 times with a solvent.<Br> | 1.Diluted DNA samples 50 times with a solvent.<Br> | ||
2.Turned on the machine; GeneQuant 100.<Br> | 2.Turned on the machine; GeneQuant 100.<Br> | ||
- | 3.Poured the solvent 100 | + | 3.Poured the solvent 100 µL into a cuvette and adjusted 0.<Br> |
4.Threw the solvent, poured the DNA sample.<Br> | 4.Threw the solvent, poured the DNA sample.<Br> | ||
5.Measured the concentration. <Br> | 5.Measured the concentration. <Br> | ||
- | + | <b>・Results</b><Br> | |
Concentration of fdh4AB was 40ng/µl.<Br> | Concentration of fdh4AB was 40ng/µl.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Ligation</b><Br> | |
- | 1.Mixed following | + | 1. Mixed following: total 5µL<Br> |
- | Vector DNA | + | (Vector DNA 1µL / Insert DNA 1.6µL / TE 2.4µL) |
- | Insert DNA | + | 2. Added Ligation Mix 5µl.<Br> |
- | TE | + | 3. Incubated at room temperature for 10 minutes.<Br> |
- | + | ||
- | + | ||
- | 2.Added Ligation Mix 5µl.<Br> | + | |
- | 3.Incubated at room temperature for 10 minutes.<Br> | + | |
<Br> | <Br> | ||
- | + | <b>・Transformation</b><Br> | |
- | 1.Melt competent cells on ice.<Br> | + | 1. Melt competent cells on ice.<Br> |
- | 2.Poured DNA solution 10µl into competent cells calmly.<Br> | + | 2. Poured DNA solution 10µl into competent cells calmly.<Br> |
- | 3.Incubated on the ice for 40 minutes.<Br> | + | 3. Incubated on the ice for 40 minutes.<Br> |
- | 4.Heat shocked at 42°C for 45 seconds.<Br> | + | 4. Heat shocked at 42°C for 45 seconds.<Br> |
- | 5.Incubated on the ice for 2-3 minutes.<Br> | + | 5. Incubated on the ice for 2-3 minutes.<Br> |
- | 6.Plated on an agar medium, and then incubated at 37°C for overnight.<Br> | + | 6. Plated on an agar medium, and then incubated at 37°C for overnight.<Br> |
<Br> | <Br> | ||
+ | <b>・Result</b> | ||
There was no colony.<Br> | There was no colony.<Br> | ||
<Br> | <Br> | ||
- | |||
- | |||
- | |||
<b>■ 5th Sep<b/></p> | <b>■ 5th Sep<b/></p> | ||
<Br><p class="description3"> | <Br><p class="description3"> | ||
- | + | <b>・Ordered P.putida arrive</b><Br> | |
- | + | (Construction of Device1)<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of no bands<Br> | |
- | + | ・Colony PCR of transformed E. coli (pfrm-frmR-GFP), electrophoresis<Br> | |
- | + | →<b>Success: 2 of 15 samples are appropriate bands!</b><Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of transformation miss<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of annealing temperature<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of annealing temperature<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →<b>Success: 1 of 15 samples is appropriate band!</b><Br> | |
- | + | ||
<Br> | <Br> | ||
- | + | <b>・Digestion</b><Br> | |
- | 1.Mixed following<Br> | + | 1. Mixed following: total 50µL<Br> |
- | + | (milliQ 5.5µl / DNA solution(fdh4AB) 40µl / 10×K buffer 2.5µl / BamHⅠ 1.0µl / SacⅠ 1.0µl<Br> | |
- | milliQ | + | 2. Incubated for 2 hours at 37°C.<Br> |
- | DNA solution | + | 3. Then, electrophoresed.<Br> |
- | 10×K buffer | + | |
- | BamHⅠ | + | |
- | + | ||
- | + | ||
<Br> | <Br> | ||
- | + | <b>・Results</b><Br> | |
- | + | ||
- | + | ||
- | + | ||
There was a thin band.<Br> | There was a thin band.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Ligation</b><Br> | |
- | 1.Mixed following .<Br> | + | 1. Mixed following: total 5.0µL<Br> |
- | Vector DNA | + | (Vector DNA 4µl / Insert DNA 0.5µl / TE 0.5µl)<Br> |
- | Insert DNA | + | |
- | TE | + | |
- | + | ||
- | <Br> | + | |
2.Added Ligation Mix 5µl.<Br> | 2.Added Ligation Mix 5µl.<Br> | ||
3.Incubated at room temperature for 10 minutes.<Br> | 3.Incubated at room temperature for 10 minutes.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Transformation<Br> | |
- | 1.Melt competent cells on ice.<Br> | + | 1. Melt competent cells on ice.<Br> |
- | 2.Poured DNA solution 10µl into competent cells calmly.<Br> | + | 2. Poured DNA solution 10µl into competent cells calmly.<Br> |
- | 3.Incubated on the ice for 40 minutes.<Br> | + | 3. Incubated on the ice for 40 minutes.<Br> |
- | 4.Heat shocked at 42°C for 45 seconds.<Br> | + | 4. Heat shocked at 42°C for 45 seconds.<Br> |
- | 5.Incubated on the ice for 2-3 minutes.<Br> | + | 5. Incubated on the ice for 2-3 minutes.<Br> |
- | 6.Plated on an agar medium, and then incubated at 37°C for overnight.<Br> | + | 6. Plated on an agar medium, and then incubated at 37°C for overnight.<Br> |
<Br> | <Br> | ||
<Br> | <Br> | ||
Line 247: | Line 214: | ||
<b>■ 6th Sep<b/></p><Br> | <b>■ 6th Sep<b/></p><Br> | ||
<p class="description3"> | <p class="description3"> | ||
- | + | (Construction of Device1)<Br> | |
- | + | <b>・Colony PCR:</b> transformed E. coli (pfrm-frmR-GFP)<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of transformation miss<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of transformation miss<Br> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<Br> | <Br> | ||
- | + | (Construction of Device3) | |
+ | <b>・Colony PCR</b> of transformed E. coli (p-fdh4AB)<Br> | ||
+ | ・Electrophoresis<Br> | ||
+ | →Failure: because of no bands<Br> | ||
+ | <b>・Electrophoresis of PCR products:</b> before digestion of fdh4AB.<Br> | ||
+ | 1. Put an agalose gel into the tank, and poured TBE buffer.<Br> | ||
+ | 2. Mixed DNA samples : loading buffer = 9 : 1. Loaded samples into wells.<Br> | ||
+ | 3. Electrophoresed, stopped when samples move 2/3.<Br> | ||
<Br> | <Br> | ||
- | + | <b>・Results</b><Br> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
There was a thin band.<Br> | There was a thin band.<Br> | ||
<Br> | <Br> | ||
<Br> | <Br> | ||
- | + | <b>・Densitometry</b><Br> | |
- | 1.Diluted DNA samples 50 times with a solvent.<Br> | + | 1. Diluted DNA samples 50 times with a solvent.<Br> |
- | 2.Turned on the machine; GeneQuant 100.<Br> | + | 2. Turned on the machine; GeneQuant 100.<Br> |
- | 3.Poured the solvent 100 l into a cuvette and adjusted 0.<Br> | + | 3. Poured the solvent 100 l into a cuvette and adjusted 0.<Br> |
- | 4.Threw the solvent, poured the DNA sample.<Br> | + | 4. Threw the solvent, poured the DNA sample.<Br> |
- | 5.Measured the concentration. <Br> | + | 5. Measured the concentration. <Br> |
<Br> | <Br> | ||
- | + | <b>・Results</b><Br> | |
Concentration of fdh4AB was 8ng/µl<Br> | Concentration of fdh4AB was 8ng/µl<Br> | ||
Line 282: | Line 248: | ||
<b>■ 7th Sep<b/></p><Br> | <b>■ 7th Sep<b/></p><Br> | ||
<p class="description3"> | <p class="description3"> | ||
- | + | (Construction of Device3)<Br> | |
- | + | <b>・PCR of insert (fdh4AB)</b><Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →<b>Success: We observed appropriate bands but a lot of smear.</b><Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →<b>Success: We observed appropriate bands!</b><Br> | |
- | + | <b>・Colony PCR:</b> transformed E. coli (p-fdh4AB)<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of PCR condition or ligation miss<Br> | |
- | + | <Br> | |
- | + | (Construction of Device2)<Br> | |
- | + | ・Preparation of culture plates for P.putida (FDH)<Br> | |
+ | ・Cultivation of P.putida (FDH) on plate<Br> | ||
<b>■ 9th Sep<b/></p><Br> | <b>■ 9th Sep<b/></p><Br> | ||
<p class="description3"> | <p class="description3"> | ||
- | + | (Construction of Device2)<Br> | |
- | + | ・PCR of insert (FDH)<Br> | |
- | + | ・Electrophoresis<Br> | |
- | + | →Failure: because of no bands<Br> | |
Latest revision as of 16:58, 26 September 2012
■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
■4th week (9. 3 - 9. 9)
■3rd September
(Construction of Device3)
・PCR of insert (fdh4AB), electrophoresis
→Failure: because of too dilute bands
・PCR of insert (fdh4AB), electrophoresis
→Failure: because of annealing temperature
・PCR of insert (fdh4AB), electrophoresis
→Success: We observed appropriate bands!
・Electrophoresis of PCR products: before digestion of fdh4AB, after PCR products
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
One sample appeared a band.
・Gel extraction
1. Excised the DNA fragment from an aglose gel. For each 100mg of agalose gel added 200µl Buffer NT.
2. Incubated sample for 5-10 minutes at 50°C. Vortexed the sample briefly every 2-3 minutes until the gel slice was completely dissolved.
3. Placed a NucleoSpin Extract ⅡColumn into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000×g. Discarded flow-through and placed the column back into the collection tube.
4. Added 700µl Buffer NT3 to the NucleoSpin Extract ⅡColumn. Centrifuged for 30 seconds at 11,000×g . Discarded flow-through and placed the column back into the collection tube.
5. Centrifuged for 1 minute at 11,000×g.
・Densitometry
1. Diluted DNA sample 50 times with a solvent.
2. Turned on the machine: GeneQuant 100
3. Poured the solvent 100µl into a cuvette and adjusted 0.
4. Threw the solvent, poured the DNA sample.
5. Measured the concentration.
・Results
Concentration of fdh4AB was 38ng/µl
・Ligation
1. Mixed following .
No.1: Total 5µL (Vector DNA 1µL / Insert DNA 1.6µL / TE 2.4µL)
No.2: Total 5µL (Vector DNA 1µL / Insert DNA 2.3µL / TE 1.7µL)
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°Cfor 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.
・Results
There was some colony.
■ 4th Sep
(Construction of Device1)
・Colony PCR of transformed E. coli (pfrm-frmR-GFP)
・Electrophoresis
→Failure: because of no bands
・Digestion
1. Mixed following
fdh4AB: total 50µL
(milliQ 5.5µL / DNA solution 40µL / 10×K buffer 2.5µL / BamH 1.0µL / SacⅠ 1.0µL
2.Incubated for 2 hours at 37°C.
・Electrophoresis
1.Put an agarose gel into the tank, and poured TBE buffer.
2.Mixed DNA sample : loading buffer = 9 : 1. Loaded sample into wells.
3.Electrophoresed, stopped when sample move 2/3.
・Results
There was no target band.
・Electrophoresis of PCR products: before digestion of fdh4AB
→diluted 10 times and 50 times of PCR products of fdh4AB.
1.Put an agarose gel into the tank, and poured TBE buffer.
2.Mixed DNA sample : loading buffer = 9 : 1. Loaded samples into wells.
3.Electrophoresed, stopped when samples move 2/3.
・Results
There was the target band.
・Gel extraction
1. Excised the DNA fragment from an agarose gel. For each 100mg of agarose gel added 300µl Buffer NT.
2. Incubated sample for 5-10 minutes at 50°C. Vortexed the sample briefly every 2-3 minutes until the gel slice was completely dissolved.
3. Placed a NucleoSpin Extract ⅡColumn into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000×g. Discarded flow-though 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 was 40ng/µl.
・Ligation
1. Mixed following: total 5µL
(Vector DNA 1µL / Insert DNA 1.6µL / TE 2.4µL)
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.
・Result
There was no colony.
■ 5th Sep
・Ordered P.putida arrive
(Construction of Device1)
・Electrophoresis
→Failure: because of no bands
・Colony PCR of transformed E. coli (pfrm-frmR-GFP), electrophoresis
→Success: 2 of 15 samples are appropriate bands!
・Electrophoresis
→Failure: because of transformation miss
・Electrophoresis
→Failure: because of annealing temperature
・Electrophoresis
→Failure: because of annealing temperature
・Electrophoresis
→Success: 1 of 15 samples is appropriate band!
・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.
3. Then, electrophoresed.
・Results
There was a thin band.
・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.
■ 6th Sep
(Construction of Device1)
・Colony PCR: transformed E. coli (pfrm-frmR-GFP)
・Electrophoresis
→Failure: because of transformation miss
・Electrophoresis
→Failure: because of transformation miss
(Construction of Device3)
・Colony PCR of transformed E. coli (p-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.
・Results
There was a thin 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 8ng/µl
■ 7th Sep
(Construction of Device3)
・PCR of insert (fdh4AB)
・Electrophoresis
→Success: We observed appropriate bands but a lot of smear.
・Electrophoresis
→Success: We observed appropriate bands!
・Colony PCR: transformed E. coli (p-fdh4AB)
・Electrophoresis
→Failure: because of PCR condition or ligation miss
(Construction of Device2)
・Preparation of culture plates for P.putida (FDH)
・Cultivation of P.putida (FDH) on plate
■ 9th Sep
(Construction of Device2)
・PCR of insert (FDH)
・Electrophoresis
→Failure: because of no bands