From 2013.igem.org
Sep 1
Transformation
No name
| 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
No name
| Sample | base pair
|
| 8/31 tRNA Spinach(pSB1C3) | 459
|
| 8/31 pT181 antisense(2)(pSB1C3)(E+S) | 415
|
| 8/31 pT181 attenuator(2)(pSB1C3)(X+P) | 601
|
| 8/31 pT181 antisense(2)(pSB1C3)(X+P) | 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
|
fig
Ligation
Nakamoto
| state | Vector | Inserter | Ligation High ver.2
|
| experiment | DT(E+S) | 5.5 | 8/28 RBS-lysis1(E+S)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&dig:C 1hour.
Liquid Culture
Tatsui,Stephane,Nakamoto
| Sample | medium
|
| 8/21 pSB1C3(BBa_J04450)(1) | Plusgrow medium(+CP)
|
| 8/21 pSB1C3(BBa_J04450)(2) | Plusgrow medium(+CP)
|
| 8/18 RBS-lysis3-DT | Plusgrow medium(+CP)
|
- at the same time made maste plate.
- incubate 37&dig:C 8hour.
M9 Medium
No name
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
Plating
Liquid Culture
"
