"
Team:Chiba/Calendar-Home/25 September 2008
From 2008.igem.org
24 September 2008 <|> 26 September 2008
Laboratory work
Team:Communication
Team:Output
colony PCR
Vector:[http://partsregistry.org/Part:BBa_R0062 BBa_R0062] Insert:[http://partsregistry.org/Part:BBa_E0240 BBa_E0240]
Vector:[http://partsregistry.org/Part:BBa_R0062 BBa_R0062]
| Sample No. | each |
| culture(μL) | 1 |
| Fwd primer(μL) | 1.5 |
| Rev primer(μL) | 1.5 |
| Taq(DNA pol)(μL) | 0.2 |
| dNTPmix(μL) | 3 |
| thermo pol Buffer(μL) | 3 |
| dH2O(μL) | 19.8 |
| TOTAL(μL) | 30 |
->95°C 5 min ->(95°C 1 min 50°C 30sec 72°C 1 min)×25 ->72°C 10 min
Digestion test
[http://partsregistry.org/Part:BBa_I6206 BBa_I6206]
[http://partsregistry.org/Part:BBa_I6101 BBa_I6101]
[http://partsregistry.org/Part:BBa_I6042 BBa_I6042]
[http://partsregistry.org/Part:BBa_I0462 BBa_I0462]
| Sample No | each |
| DNA(μL) | 2 |
| EcoRI(μL) | 0.2 |
| EcoRI Buffer(μL) | 1 |
| dH2O(μL) | 7 |
| TOTAL(μL) | 30 |
->30 min 37°C
gelcheck
[http://partsregistry.org/Part:BBa_I6206 BBa_I6206](digestion product)
[http://partsregistry.org/Part:BBa_I6101 BBa_I6101](digestion product)
[http://partsregistry.org/Part:BBa_I6042 BBa_I6042](digestion product)
[http://partsregistry.org/Part:BBa_I0462 BBa_I0462](digestion product)
[http://partsregistry.org/Part:BBa_I0642 BBa_I0642](natural)
| Sample No | each(digestion) | I0462(natural) |
| DNA(μL) | 10 | 1 |
| Loading Dye(μL) | 2 | 1 |
| dH2O(μL) | - | 4 |
| TOTAL(μL) | 12 | 6 |
digestion
[http://partsregistry.org/Part:BBa_I0462 BBa_I0462]
| DNA(μL) | 25 |
| XbaI(μL) | 1 |
| PstI | 1 |
| Buffer 3(μL) | 4 |
| BSA(μL) | 4 |
| dH2O(μL) | 5 |
| TOTAL(μL) | 40 |
->3 hours 37°C
->Zymo ->10μL
Ligation
Vector:[http://partsregistry.org/Part:BBa_I7100 BBa_I7100]
Insert:[http://partsregistry.org/Part:BBa_R0062 BBa_R0062]
Insert:[http://partsregistry.org/Part:BBa_I0462 BBa_I0462]
| I7100 | 1.3 | 1.3 |
| [http://partsregistry.org/Part:BBa_R0062 BBa_R0062] | 2 | - |
| [http://partsregistry.org/Part:BBa_I0462 BBa_I0462] | 3.3 | - |
| Ligase(μL) | 1 | 1 |
| Ligase Buffer(μL) | 2 | 2 |
| dH2O(μL) | 0.4 | 5 |
| TOTAL(μL) | 10 | 10 |
->3 hours 37°C
->Transformation
