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Template:Kyoto/Notebook/Sep 1
From 2013.igem.org
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|8/31 tRNA Spinach(pSB1C3)||459 | |8/31 tRNA Spinach(pSB1C3)||459 | ||
|- | |- | ||
| - | |8/31 pT181 antisense(2)(pSB1C3)( | + | |8/31 pT181 antisense(2)(pSB1C3)(EcoRI+SpeI)||415 |
|- | |- | ||
| - | |8/31 pT181 attenuator(2)(pSB1C3)( | + | |8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI)||601 |
|- | |- | ||
| - | |8/31 pT181 antisense(2)(pSB1C3)( | + | |8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI)||415 |
|- | |- | ||
|8/31 pT181 attenuator(2)-DT||754 | |8/31 pT181 attenuator(2)-DT||754 | ||
| Line 61: | Line 61: | ||
|5min||30s||30s||30min||30cycle | |5min||30s||30s||30min||30cycle | ||
|} | |} | ||
| - | [[ | + | [[File:Igku Sep1 ligation production PCR.jpg]] |
</div> | </div> | ||
| + | |||
===Ligation=== | ===Ligation=== | ||
<div class="experiment"> | <div class="experiment"> | ||
<span class="author">Nakamoto</span> | <span class="author">Nakamoto</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | !state||colspan="2"|Vector||colspan="2"|Inserter | + | !state||colspan="2"|Vector||colspan="2"|Inserter |
|- | |- | ||
| - | |experiment||DT( | + | |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.<br> | 1. Samples were evaporeted used evaporator into about 7 µL.<br> | ||
| - | 2. Add Ligation High 3.5µ:L and incubate 16& | + | 2. Add Ligation High 3.5µ:L and incubate 16°C 1hour.<br> |
</div> | </div> | ||
| Line 81: | Line 82: | ||
!Sample||medium | !Sample||medium | ||
|- | |- | ||
| - | |8/21 pSB1C3(BBa_J04450)(1)||Plusgrow medium(+CP) | + | |8/21 pSB1C3(BBa_J04450)-(1)||Plusgrow medium(+CP) |
|- | |- | ||
| - | |8/21 pSB1C3(BBa_J04450)(2)||Plusgrow medium(+CP) | + | |8/21 pSB1C3(BBa_J04450)-(2)||Plusgrow medium(+CP) |
|- | |- | ||
| - | | | + | |RBS-lysis3-DT||Plusgrow medium(+CP) |
|} | |} | ||
| - | + | ||
| - | + | ||
| - | + | ||
===M9 Medium=== | ===M9 Medium=== | ||
| Line 139: | Line 138: | ||
<div class="experiment"> | <div class="experiment"> | ||
<span class="author">Hirano</span> | <span class="author">Hirano</span> | ||
| - | Apply entA | + | Apply entA-colony liquid culture (100µL) on M9(Km)plate and incubate 37°C</div> |
| - | + | 2 fold diluted OD 600 2.107 | |
===Liquid Culture=== | ===Liquid Culture=== | ||
<div class="experiment"> | <div class="experiment"> | ||
| + | <span class="author">Hirano</span> | ||
| + | {| class="wikitable" | ||
| + | !Sample||medium | ||
| + | |- | ||
| + | |9/1 entA- Master Plate-(4)||SOB Medium(+Km) | ||
| + | |} | ||
</div> | </div> | ||
Latest revision as of 17:19, 25 September 2013
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) |
