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Template:Kyoto/Notebook/Sep 1
From 2013.igem.org
Contents |
Sep 1
Transformation
| Name | Sample | Competent Cells | Total | Plate |
|---|---|---|---|---|
| 8/31 tRNA-Spinach(pSB1C3) | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 antisense(pSB1C3) | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 attenuator(pSB1C3) | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 antisense(pSB1C3) | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 attenuator(2)-DT | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 antisense-DT | 2µL | 20µL | 22µL | CP |
| 8/31 tRNA-Spniach-DT | 2µL | 20µL | 22µL | CP |
| 8/31 RBS-lysis2-DT | 2µL | 20µL | 22µL | CP |
| 8/31 Pconst-RBS-tetR-DT | 2µL | 20µL | 22µL | Amp |
| 8/31 Plac-RBS-lacZα-DT | 2µL | 20µL | 22µL | CP |
| 8/31 Ptet-RBS-lacZα-DT | 2µL | 20µL | 22µL | CP |
| 8/31 | 2µL | 20µL | 22µL | CP |
ligation production PCR
| Sample | base pair |
|---|---|
| 8/31 tRNA Spinach(pSB1C3) | 459 |
| 8/31 pT181 antisense(2)(pSB1C3)(EcoRI+SpeI) | 415 |
| 8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI) | 601 |
| 8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI) | 415 |
| 8/31 pT181 attenuator(2)-DT | 754 |
| 8/31 pT181 antisense(2)-DT | 577 |
| 8/31 tRNA Spinach-DT | 596 |
| 8/22 pSB1C3(2)(controll) | 1383 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30min | 30cycle |
Ligation
| state | Vector | Inserter | ||
|---|---|---|---|---|
| experiment | DT(EcoRI+SpeI) | 5.5 | 8/28 RBS-lysis1(EcoRI+SpeI)2.4ng/µL | 2.4 |
1. Samples were evaporeted used evaporator into about 7 µL.
2. Add Ligation High 3.5µ:L and incubate 16°C 1hour.
Liquid Culture
| Sample | medium |
|---|---|
| 8/21 pSB1C3(BBa_J04450)-(1) | Plusgrow medium(+CP) |
| 8/21 pSB1C3(BBa_J04450)-(2) | Plusgrow medium(+CP) |
| RBS-lysis3-DT | Plusgrow medium(+CP) |
M9 Medium
5xM9 liquid medium
| volume | 100mL |
|---|---|
| NaHPO4 | 30g |
| KH2PO | 3g |
| Nacl | 0.25g |
| NH4Cl | 0.5g |
| 3.75% agar solution | 400mL |
| 1M MgSO4 | 0.5mL |
| 2M Glucose | 2.8mL |
| 1% Vitamin B1 | 0.5mL |
| 1M CaCl2 | 0.05mL |
1. mix 5xM9 medium and 3.75%Agar solution 400mL
2. Add 1M MgSO4, 2M Glucose and 1 VitaminB1 0.5mL
3. Add CaCl2 while shaking Erlenmeyer flask.
Miniprep
| DNA | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| 8/21 pSB1C3(1) | 309.2 | 1.69 | 1.72 |
| 8/21 pSB1C3(2) | 201.6 | 1.65 | 1.76 |
Kanamycin applicaton
Add 20µL Kanamycin into 80µL MilliQ and apply it on a 9/1 M9 plate
Plating
Apply entA-colony liquid culture (100µL) on M9(Km)plate and incubate 37°C
2 fold diluted OD 600 2.107
Liquid Culture
| Sample | medium |
|---|---|
| 9/1 entA- Master Plate-(4) | SOB Medium(+Km) |
