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

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<B>■Experiment</B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/1st_week_(8.13_-_8.19)">1st week (8.13 - 8.19)</A></B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/2nd_week_(8.20_-_8.26)">2nd week (8.20 - 8.26)</A></B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/3rd_week_(8.27_-_9._2)">3rd week (8.27 - 9. 2)</A></B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/4th_week_(9._3_-_9._9)">4th week (9. 3 - 9. 9)</A></B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/5th_week_(9.10_-_9.16)">5th week (9.10 - 9.16)</A></B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/6th_week_(9.17_-_9.23)">6th week (9.17 - 9.23)</A></B><Br>
<B><A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Experiment/7th_week_(9.24_-_9.30)">7th week (9.24 - 9.30)</A></B><Br>
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<Br>Experiment<Br>
<Hr><Br>
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<p class="description">
<b>■5th week (9.10 - 9.16)</b><Br></p>
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<b>■10th Sep</b><Br>
(Construction of Device2)<Br>
・PCR of insert (FDH)<Br>
・Electrophoresis<Br>
→Failure: because of a lot of smear<Br>
<b>・Ordered Device1 and Device2 plasmids arrive!</b><Br>
<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>
<B>・Results</B><Br>
There was the target band.<Br>
<Br>
<b>・Densitometry</b><Br>
1. Diluted DNA samples 50 times with a solvent.<Br>
2. Turned on the machine; GeneQuant 100.<Br>
3. Poured the solvent 100 µl into a cuvette and adjusted 0.<Br>
4. Threw the solvent, poured the DNA sample.<Br>
5. Measured the concentration. <Br>
<Br>
<b>・Results</b><Br>
Concentration of fdh4AB: 20ng/µl.<Br>
<Br>
<b>・Digestion</b><Br>
1. Mixed following: total 50µL<Br>
(milliQ 5.5µl / DNA solution(fdh4AB) 40µ / 10×K buffer 2.5µl / BamHⅠ 1.0µl / SacⅠ 1.0µl<Br>
2. Incubated for 2 hours at 37°C.<Br>
<Br>
<b>・Gel extraction</b><Br>
<b>・Densitometry</b><Br>
Concentration of fdh4AB: 2ng/µl.<Br>
<Br>
<b>・Ligation</b><Br>
1. Mixed following: total 5.0µl<Br> 
(Vector DNA 4µl / Insert DNA 0.5µl / TE 0.5µl<Br>
2. Added Ligation Mix 5µl.<Br>
3. Incubated at room temperature for 10 minutes.<Br>
<Br>
<b>Transformation</b><Br>
1. Melt competent cells on ice.<Br>
2. Poured DNA solution 10µl into competent cells calmly.<Br>
3. Incubated on the ice for 40 minutes.<Br>
4. Heat shocked at 42°C for 45 seconds.<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>
<Br>
<Br>
<b>■11th Sep</b><br /><Br>
(Construction of Device3)<Br>
・PCR of insert (fdh4AB)<Br>
・Electrophoresis<Br>
→Failure: because of no bands<Br>
<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>
<b>・Gel extraction of fdh4AB</b>
1. Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.<Br>
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.<Br>
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.<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 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.<Br>


<Br>
<b>・Results</b><Br>
There was the target band.<Br>
<Br>
<b>・Densitometry</b><Br>
1. Diluted DNA samples 50 times with a solvent.<Br>
2. Turned on the machine; GeneQuant 100.<Br>
3. Poured the solvent 100 µl into a cuvette and adjusted 0.<Br>
4. Threw the solvent, poured the DNA sample.<Br>
5. Measured the concentration. <Br>
<Br>
<b>・Results</b><Br>
Concentration of fdh4AB: 20ng/µl.<Br>
<Br>
<b>・Digestion</b><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>
2.Incubated for 2 hours at 37°C.<Br>
<Br>
<b>・Gel extraction</b><Br>
<b>・Densitometry</b><Br>
Concentration of fdh4AB: 2ng/µl.<Br>
<Br>
<b>・Ligation</b><Br>
1. Mixed following: total 5.0µl<Br>
(Vector DNA 4µl / Insert DNA 0.5µl / TE 0.5µl)<Br>
2. Added Ligation Mix 5µl.<Br>
3. Incubated at room temperature for 10 minutes.<Br>

<b>・Transformation</b><Br>
1. Melt competent cells on ice.<Br>
2. Poured DNA solution 10µl into competent cells calmly.<Br>
3. Incubated on the ice for 40 minutes.<Br>
4. Heat shocked at 42°C for 45 seconds.<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>
<Br>
<Br>
<b>■12th Sep</b><br /><Br>
<Br>
(Construction of Device3)<Br>
<b>・Colony PCR:</b> transformed E. coli (p-fdh4AB)<Br>
・Electrophoresis<Br>
→Failure: because of transformation miss<Br>
・Electrophoresis<Br>
→Failure: because of transformation miss<Br>
・Electrophoresis<Br>
→Failure: because of transformation miss<Br>
・Electrophoresis<Br>
→Failure: because of transformation miss<Br>
<Br>
<Br>
<b>■13th Sep</b><br /><Br>
(Construction of Device3)<Br>
・PCR of insert (fdh4AB)<Br>
・Electrophoresis<Br>
→<b>Success:</b> we observed appropriate bands but a lot of smear<Br>
・<b>Colony PCR:</b> transformed E. coli (p-fdh4AB)<Br>
・Electrophoresis<Br>
→Failure: because of no appropriate bands<Br>
<Br>
<b>Transformation</b><br>
1. Melt competent cells on ice.<Br>
2. Poured DNA solution (pSB1K3) 10 µl into competent cells calmly<Br>
3. Incubated on the ice for 40 minutes<Br>
4. Heat shocked at 42 °C for 45 seconds<Br>
5. Incubated on the ice for 2-3 minutes<Br>
6. Added 600 µl LB medium, and then incubated for 30 minutes at 37 °C<br>
7. Plated on an agar medium, and then incubated at 37 °C for overnight<Br>



<Br>
<Br>
<b>■14th Sep</b><br />
<b>New fdh4AB primers arrive</b><Br>
(Construction of Device3)<Br>
・PCR of insert (fdh4AB)<Br>
・Electrophoresis<Br>
→Failure: because of artificial mistake?<Br>
・PCR of insert (fdh4AB)<Br>
・Electrophoresis<Br>
→Success: appropriate bands are clearer than before!<Br>
<Br>
<b>・Gel extraction</b><Br>
1. Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.<Br>
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.<Br>
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.<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 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.<Br>
<Br>
<b>・Densitometry</b><Br>
1. Diluted DNA samples 50 times with a solvent.<Br>
2. Turned on the machine; GeneQuant 100.<Br>
3. Poured the solvent 100 μl into a cuvette and adjusted 0.<Br>
4. Threw the solvent, poured the DNA sample.<Br>
5. Measured the concentration.<Br>
<Br>
<b>・Results</b><Br>
Concentration of fdh4AB no.1: 8 ng/ µl, no.2: 13 ng/ µl.<Br>
<Br>
<b>・Digestion</b><Br>
1. Mixed following: total 50µl<Br>
(milliQ 7.5 µl / DNA solution 38 µl / 0.5x K buffer 2.5 µl / SacⅠ 1 µl / BamHⅠ 1 µl)<Br>
<Br>
2. Incubated at 37 °C for 2 hours.<Br>
3. Heat inactivated for 20 minutes at 50 °C.<Br>
<Br>
<Br>
<b>■15th Sep</b><Br>
<b>・Gel-extracted fdh4AB band part PCR</b>
The densitometry result: DNA No.14･･･40μg/μl, DNA No.16…8μg/μl<Br>
・ Made PCR solution<Br>
tube4 ×4set 50μl ( DW 140.5 / Buffer 42.5 / dNTP 17 /pF 3.1875 / pR 3.1875 /DNA 4 / Poly 2.125)<Br>
10 fold dilution of DNA (DW: DNA=18:2)<Br>
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)<Br>
・ PCR with Thermal Cycler: (98℃: 5min, 98℃: 10sec, 63℃: 5sec, 72℃: 2min40sec, 72℃: 5min, 4℃: ∞)<Br>
・ Electrophoresis of PCR product<Br>
<b>・ Result</b><Br>
PCR product was seen only on undiluted DNA No.14 but it was smearing<Br>
<Br>
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