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Template:Kyoto/Notebook/Sep 16
From 2013.igem.org
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<span class="author">じっけんしゃ</span> | <span class="author">じっけんしゃ</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | ! ||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|| | + | |2 cuts||15.8||0||0||1||1||3||3||6.2||30 |
|- | |- | ||
| - | |NC|| | + | |NC||0.8||0||0||0||0||1||1||7.2||10 |
|} | |} | ||
| + | {| class="wikitable" | ||
| + | ! ||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 | ||
| + | |} | ||
| + | {| class="wikitable" | ||
| + | ! ||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 | ||
| + | |} | ||
| + | {| class="wikitable" | ||
| + | ! ||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 | ||
| + | |} | ||
| + | {| class="wikitable" | ||
| + | ! ||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=== | ===Ligation=== | ||
Revision as of 06:33, 24 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 |
| 9/14Plac332.5 | 1.51 | 2.33 | |
| Ptet-RBS-laczα-DT | 231.3 | 1.84 | 1.57 |
PCR
| 9/6pT181 anti psB1C3(1) | KOD plus | 10x buffer | dNTP | MgSO4 | GG1_1C3_fwd primer | GG2_1C3_rev primer | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 57°C | 68°C | 30cycles |
| 9/6 12-1R aptamer-DT | KOD plus | 10x buffer | dNTP | MgSO4 | GG1_1C3_fwd primer | GG1_1C3_rev primer | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 57°C | 68°C | 30cycles |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 100bp ladder | ||
| 2 | anti | ||
| 3 | anti | ||
| 4 | anti | ||
| 5 | tet aptamer-DT | ||
| 6 | tet aptamer-DT | ||
| 7 | tet aptamer-DT | ||
| 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 | name | xx µg/mL | name | xx µg/mL | xx µg/mL |
| NC | name | xx µg/mL | name | xx µg/mL | xx µg/mL |
PCR
| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | SasA_fwd primer | SasA_rev primer | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| °C | °C | °C | °C | -- |
| （◎Д◎） |
