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Template:Kyoto/Notebook/Sep 16
From 2013.igem.org
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|experiment||Pcon-pT181anti-spin-DT(X+P)||5.0µg/mL||pSB4K5(X+P)||18.1µg/mL||xx µg/mL | |experiment||Pcon-pT181anti-spin-DT(X+P)||5.0µg/mL||pSB4K5(X+P)||18.1µg/mL||xx µg/mL | ||
|- | |- | ||
| - | |experiment||pC181 | + | |experiment||pC181 attenuator(X+P)||8.2µg/mL||pSB1C3(X+P)||26.0µg/mL||xx µg/mL |
|} | |} | ||
Revision as of 16:24, 25 September 2013
Contents |
Sep 16
Liquid Culture
| Sample | medium |
|---|---|
| pSB1C3+P-KaiBC① | Plusgrow medium(+CP) |
| pSB1C3+P-KaiBC② | Plusgrow medium(+CP) |
Colony PCR
| Sample | base pair |
|---|---|
| pSB1C3+P-KaiBC① | 283 |
| pSB1C3+P-KaiBC② | 283 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 2min | 30s | 30s | 18s | 30cycles |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kb ladder | |
| 2 | pT181 attenuator | X+P |
| 3 | pT181 attenuator | X+P |
| 4 | pT181 attenuator | X+P |
| 6 | pSB1C3 | E+S |
| 7 | pSB1C3 | E+S |
| 8 | pSB1C3 | E+S |
| 10 | pSB4K5 | X+P |
| 11 | pSB4K5 | X+P |
| 12 | pSB4K5 | X+P |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pT181 attenuator(X+P) | 8.2 | 1.62 | 0.11 |
| pSB1C3(E+S) | 49.5 | 1.48 | 0.18 |
| pSB4K5(X+P) | 18.1 | 1.73 | 0.57 |
| DT(S+P) | 70.4 | 1.36 | 1.02 |
| aptamer12-1R-DT(S+P) | 42.0 | 1.82 | 0.47 |
| spinach-DT(S+P) | 36.8 | 1.79 | 0.61 |
Miniprep
| DNA | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| Dx-lux1 | 136.7 | 1.46 | 1.28 |
| RBS-laczα-DT | 237.6 | 1.88 | 1.70 |
| pSB4K5 | 148.4 | 1.55 | 1.09 |
| Pcon-KBS-tetR-DT | 126.6 | 1.73 | 1.36 |
| 9/11Plac1 | 160.0 | 1.78 | 1.28 |
| 9/15Plac2 | 144.0 | 1.73 | 1.81 |
| 9/14Plac | 332.5 | 1.51 | 2.33 |
| Ptet-RBS-laczα-DT | 231.3 | 1.84 | 1.57 |
PCR
| DNA | 9/6pT181 anti psB1C3(1) | KOD plus | 10x buffer | dNTP | MgSO4 | GG1_1C3_fwd primer | GG2_1C3_rev primer | MilliQ | total |
|---|---|---|---|---|---|---|---|---|---|
| 400pg | 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
| 800pg | 2 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 14.5 | 25 |
| 1000pg | 2.5 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 14 | 25 |
| nega | 0 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.5 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |
| DNA | 9/6 12-1R aptamer-DT | KOD plus | 10x buffer | dNTP | MgSO4 | GG1_1C3_fwd primer | GG1_1C3_rev primer | MilliQ | total |
|---|---|---|---|---|---|---|---|---|---|
| 400pg | 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
| 800pg | 2 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 14.5 | 25 |
| 1000pg | 2.5 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 14 | 25 |
| nega | 0 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.5 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |
Electrophoresis
| Lane | Sample |
|---|---|
| 1 | 100bp ladder |
| 2 | anti 1000pg |
| 3 | anti 800pg |
| 4 | anti 400pg |
| 5 | tet aptamer-DT 1000pg |
| 6 | tet aptamer-DT 800pg |
| 7 | tet aptamer-DT 400pg |
| 8 | nega |
Restriction Enzyme Digestion
| 9/16 Pcon-RBS-tetR-DT(µL) | E(µL) | S(µL) | X(µL) | P(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 15.8 | 0 | 0 | 1 | 1 | 3 | 3 | 6.2 | 30 |
| NC | 0.8 | 0 | 0 | 0 | 0 | 1 | 1 | 7.2 | 10 |
| 9/16 pSB4K5(µL) | E(µL) | S(µL) | X(µL) | P(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 13.5 | 0 | 0 | 1 | 1 | 3 | 3 | 8.5 | 30 |
| NC | 0.7 | 0 | 0 | 0 | 0 | 1 | 1 | 7.3 | 10 |
| 8/22 pSB1C3(µL) | E(µL) | S(µL) | X(µL) | P(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 18.5 | 1 | 0 | 0 | 1 | 3 | 3 | 3.5 | 30 |
| NC | 0.9 | 0 | 0 | 0 | 0 | 1 | 1 | 7.1 | 10 |
| 8/22 pSB1C3(µL) | E(µL) | S(µL) | X(µL) | P(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 18.5 | 1 | 1 | 0 | 0 | 3 | 3 | 3.5 | 30 |
| NC | 0.9 | 0 | 0 | 0 | 0 | 1 | 1 | 7.1 | 10 |
| 8/21 pT181 attenuator2(µL) | E(µL) | S(µL) | X(µL) | P(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 7.4 | 1 | 1 | 0 | 0 | 3 | 3 | 14.6 | 30 |
| NC | 0.4 | 0 | 0 | 0 | 0 | 1 | 1 | 7.6 | 10 |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | Pcon-pT181anti-spin-DT(X+P) | 5.0µg/mL | pSB4K5(X+P) | 18.1µg/mL | xx µg/mL |
| experiment | pC181 attenuator(X+P) | 8.2µg/mL | pSB1C3(X+P) | 26.0µg/mL | xx µg/mL |
