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Team:Chiba/jk/γ/week 6
From 2008.igem.org
Contents |
WEEK 6
22,September,2008
Time delay
23,September,2008
Digestion test
BBa_E0240
| Sample No | E0240 |
| DNA | 2 |
| EcoRⅠ | 0.5 |
| PstⅠ | 0.5 |
| BSA(x10) | 1 |
| EcoRⅠ Buffer(x10) | 1 |
| dH2O | 5 |
| TOTAL | 10 |
TRANSFORMATION
BBa_I6107
BBa_I6206
BBa_I6101
BBa_I6042
BBa_I0462
Digestion
Vector:BBa_R0062①
Insert:BBa_E0240②
Vector:BBa_I7100③
Insert:BBa_R0062④
Insert:BBa_J37033⑤
| Sample No | ① | ② | ③ | ④ | ⑤ |
| DNA | 25 | 25 | 25 | 25 | 25 |
| EcoRⅠ | - | - | 1 | 1 | - |
| SpeⅠ | 1 | - | 1 | 1 | - |
| PstⅠ | 1 | 1 | 1 | - | 1 |
| XbaⅠ | - | 1 | - | - | 1 | BSA(x10) | 4 | 4 | 4 | - | 4 |
| EcoRⅠ Buffer(x10) | - | - | 4 | 4 | - |
| Buffer 2 | 4 | - | - | - | - |
| Buffer 3 | - | 4 | - | - | 4 |
| dH2O | 5 | 5 | 5 | 9 | 5 |
| TOTAL | 40 | 40 | 40 | 40 | 40 |
->1hour 37℃
->SAP 1μl(Vector only) ->1hour 37℃ ->30min 65℃
->Zymo ->10μl
Gel cheack
| Sample No | each |
| DNA | 1 |
| Loading Dye | 1 |
| dH2O | 4 |
| TOTAL | 10 |
->③&④&⑤のLigationはしない
24,September,2008
Ligation
Vector:BBa_R0062①
Insert:BBa_E0240②
| Sample No | ①&② | ① |
| DNA(vector) | 1 | 1 |
| DNA(insert) | 3.2 | - |
| Ligase | 1 | 1 |
| Ligase Buffer | 2 | 2 |
| dH2O | 2.5 | 6 |
| TOTAL | 10 | 10 |
->TRANSFORMATION
25,September,2008
colony PCR
Vector:BBa_R0062 Insert:BBa_E0240
Vector:BBa_R0062
| Sample No | each |
| culture | 1 |
| Fwd primer | 1.5 |
| Rev primer | 1.5 |
| Taq(DNA pol) | 0.2 |
| dNTPmix | 3 |
| thermo pol Buffer | 3 |
| dH2O | 19.8 |
| TOTAL | 30 |
->95℃ 5min ->(95℃ 1min 50℃ 30sec 72℃ 1min)x25 ->72℃ 10min
Digestion Test
BBa_I6206
BBa_I6101
BBa_I6042
BBa_I0462
| Sample No | each |
| DNA | 2 |
| EcoRⅠ | 0.2 |
| EcoRⅠ Buffer | 1 |
| dH2O | 7 |
| TOTAL | 30 |
->30min 37℃
Gel cheack
Vector:BBa_R0062
| Sample No | each(digestion) | I0462 |
| DNA | 10 | 1 |
| Loading Dye | 2 | 1 |
| dH2O | - | 4 |
| TOTAL | 12 | 6 |
Digestion
BBa_I0462
| DNA | 25 |
| XbaⅠ | 1 |
| PstⅠ | 1 |
| Buffer 3 | 4 |
| BSA | 4 |
| dH2O | 5 |
| TOTAL | 40 |
->3hour 37℃
->Zymo ->10μl
Ligation
Vector:BBa_I7100
Insert:BBa_R0062
Insert:BBa_I0462
| I7100 | 1.3 | 1.3 |
| R0062 | 2 | - |
| I0462 | 3.3 | - |
| Ligase | 1 | 1 |
| Ligase Buffer | 2 | 2 |
| dH2O | 0.4 | 5 |
| TOTAL | 10 | 10 |
->3hour 37℃
->Transformation
26,September,2008
TIME DELAY TEST
colony PCR
Ligation product(26,September)x7
Vector:BBa_R0062
| Sample No | each |
| culture | 1 |
| Fwd primer | 1.5 |
| Rev primer | 1.5 |
| Taq(DNA pol) | 0.2 |
| dNTPmix | 3 |
| thermo pol Buffer | 3 |
| dH2O | 19.8 |
| TOTAL | 30 |
->95℃ 5min ->(95℃ 1min 50℃ 30sec 72℃ 1min)x25 ->72℃ 10min
->Gel cheack
->MINI Prep
27,September,2008
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