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Template:Kyoto/Notebook/Sep 3
From 2013.igem.org
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| + | ===Electrophoresis=== | ||
| + | |||
| + | <div class="experiment"> | ||
| + | <span class="author">Kozima</span> | ||
| + | {| class="wikitable" | ||
| + | !Lane||Sample | ||
| + | |- | ||
| + | |1||100bp ladder | ||
| + | |- | ||
| + | |2||9/1 pT181antisense(pSB1C3) | ||
| + | |- | ||
| + | |3||9/1 pT181antisense | ||
| + | |} | ||
| + | [[File:Igku Sep3 Electrophoresis(N1).jpg]]<br> | ||
</div> | </div> | ||
Revision as of 13:01, 25 September 2013
Contents |
Sep 3
Electrophoresis
| Lane | Sample |
|---|---|
| 1 | 100bp ladder |
| 2 | pT181 attenuater(2)-DT2(738) |
| 3 | Ptet-RBS-lacZα-DT-2(756) |
| 4 | Ptet-RBS-lacZα-DT-3 |
| 5 | Ptet-RBS-lacZα-DT-4 |
| 6 | Ptet-RBS-lacZα-DT-5 |
| 7 | Ptet-RBS-lacZα-DT-6 |
| 8 | RBS-lysis2-DT-2(985) |
| 9 | RBS-lysis2-DT-3 |
| 10 | 100bp ladder |
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Miniprep
| DNA | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| 9/2 14:00 pT181 attenuator | 418.4 | 1.82 | 1.80 |
| 9/2 14:00 pT181 antisense | 362.2 | 1.92 | 1.96 |
| 9/2 14:00 Spinach | 438.3 | 1.86 | 1.99 |
| 9/2 14:00 tetR-apt 12_1R | 567.5 | 1.90 | 1.99 |
| 9/2 21:00 pT181 attenuater | 320.9 | 1.90 | 2.15 |
| 9/2 21:00 Spinach | 457.0 | 1.88 | 2.24 |
Liquid Culture
| Sample | medium |
|---|---|
| Spinach-DT(9/2 master plate) | plusgrow(CP)4ml |
Restriction Enzyme Digestion
| DNA | EcoR1 | Spe1 | Xba1 | Pst1 | 10xBuffer B(EcoR1&Spe1) | 10xBuffer D(Xba1&Pst1) | 100xBSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|---|
| 9/3 pT181attenuater 14:00 418.4ng/µL | 4.8µL | 1.0µL | 1.0µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 19.9µL | 30µL |
| 9/3 pT181attenuater 14:00 418.4ng/µL | 0.2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.7µL | 10µL |
| 9/3 pT181attenuater 14:00 418.4ng/µL | 4.8µL | 0µL | 0µL | 1.0µL | 1.0µL | 0µL | 3µL | 0.3µL | 19.9µL | 30µL |
| 9/3 pT181attenuater 14:00 418.4ng/µL | 0.2µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.7µL | 10µL |
| DNA | EcoR1 | Spe1 | Xba1 | Pst1 | 10xBuffer B(EcoR1&Spe1) | 10xBuffer D(Xba1&Pst1) | 100xBSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|---|
| 9/3 pT181antisense 14:00 362.2ng/µL | 5.5µL | 1.0µL | 1.0µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 19.2µL | 30µL |
| 9/3 pT181attenuater 14:00 418.4ng/µL | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.6µL | 10µL |
| 9/3 pT181attenuater 14:00 418.4ng/µL | 5.5µL | 0µL | 0µL | 1.0µL | 1.0µL | 0µL | 3µL | 0.3µL | 19.2µL | 30µL |
| 9/3 pT181attenuater 14:00 418.4ng/µL | 0.3µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |
| DNA | EcoR1 | Spe1 | 10xBuffer B | 100xBSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 9/3 apt 12_1R 14:00 507.5ng/µL | 3.9µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 20.8µL | 30µL |
| 9/3 apt 12_1R 14:00 507.5ng/µL | 0.2µL | 0µL | 0µL | 1µL | 0.1µL | 8.7µL | 10µL |
| DNA | EcoR1 | Xba1 | 10xBuffer D | 100xBSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 8/29 DT 166.2ng/µL | 12µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 12.7µL | 30µL |
| 8/29 DT 166.2ng/µL | 0.6µL | 0µL | 0µL | 1µL | 0.1µL | 8.3µL | 10µL |
| DNA | EcoR1 | Spe1 | 10xBuffer B | 100xBSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 8/22 RBS-lysis1(2) 96ng/µL | 21µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 3.7µL | 30µL |
| 8/22 RBS-lysis1(2) 96ng/µL | 1.0µL | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL |
| DNA | EcoR1 | Spe1 | 10xBuffer B | 100xBSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 8/30 RBS-lysis2 220ng/µL | 9.1µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 15.6µL | 30µL |
| 8/30 RBS-lysis2 220ng/µL | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL |
| DNA | EcoR1 | Spe1 | 10xBuffer B | 100xBSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 8/20 RBS-lysis3(1) 282ng/µL | 7.1µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 17.6µL | 30µL |
| 8/20 RBS-lysis3(1) 282ng/µL | 0.4µL | 0µL | 0µL | 1µL | 0.1µL | 8.5µL | 10µL |
PCR
| Plac(1) plasmid 153.7ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer (KaiA_BsaI_Mut_fwd) | primer (KaiA_BsaI_Mut_rer) | KOD plus-ver.2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.13 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16.37 | 25 |
| KaiB (plasmid) 20.2ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer (GG2_RBS_KaiB_fwd) | primer (KaiB_GG3_rer) | KOD plus-ver.2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |
| KaiC (plasmid) 4.4ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |
| DT-1 (plasmid) 153.7ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16.4 | 25 |
| PKaiBC(PCR product) 16.3ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.7 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.8 | 25 |
| SasA(PCR product) 21.6ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16 | 25 |
| RpaA(PCR product) 15.4ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.6 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.9 | 25 |
| RpaB(PCR product) 26.7ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.4 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16.1 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 68°C | -- | |
| 5min | 10sec | 30sec | 30 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 68°C | -- | |
| 5min | 10sec | 30sec | 30 |
Mutatation PCR
| KaiABC(12.6pg/µL) | Primer(Kai_BsaIMut_fwd) | Primer(Kai_BsaIMut_rer) | Prime STAR Max Premix | MilliQ | total |
|---|---|---|---|---|---|
| 1 | 1 | 1 | 25 | 22 | 50 |
| Primer(Kai_BsaIMut_fwd) | Primer(Kai_BsaIMut_rer) | Prime STAR Max Premix | MilliQ | total | |
|---|---|---|---|---|---|
| 1 | 1 | 25 | 23 | 50 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 65°C | 72°C | -- |
| 5min | 10sec | 15sec | 40sec | 30 |
Colony PCR
| Sample | base pair |
|---|---|
| 9/1 pT181antisense(pSB1C3) | 400bp |
| 9/1 pT181antisense-DT | 547bp |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5m | 30s | 30s | 30s | 30 |
Electrophoresis
| Lane | Sample |
|---|---|
| 1 | 100bp ladder |
| 2 | 9/1 pT181antisense(pSB1C3) |
| 3 | 9/1 pT181antisense |
