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Team:HokkaidoU Japan/Notebook/September17
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*Construction of GFP marker for a part which will be secreted using T3SS | *Construction of GFP marker for a part which will be secreted using T3SS | ||
*Ordered primers for construction for same part | *Ordered primers for construction for same part | ||
Revision as of 07:41, 1 October 2010
since 13 Jul 2010
since 1 Aug 2010
- Construction of GFP marker for a part which will be secreted using T3SS
- Ordered primers for construction for same part
Digestion of GFP and Double Terminator
Parts Information
| Description | BioBrick No. | Well No. | Length | Plasmid |
|---|---|---|---|---|
| GFP | BBa_E0040 | 1-14K | 720bp | pSB1A3 |
| double terminator | BBa_B0015 | 1-23L | 129bp | pSB1AK3 |
| pSB1A3 | pSB1A3 | 2157bp | pSB1A3 |
Parts in wells 1-14K and pSB1A3 were purified with mycrocon. Part 1-23L was extracted from a gel previously.
- Performed electrophoresis of 1-14K and 1-23L to estimate concentration of each solution.
- Estimated concentration from photo of electrophoresis
- pSB1A3 solution was done by other person.
- Made digestion recipes(below) based on estimated concentrations
- Made 30ul of pSB1A3 solution, but latter found it insufficient to ligate parts
- made more 50ul of it after
| Part Well No. | Amount |
|---|---|
| 1-14K | 200 ng/ul |
| 1-23L | 120 ng/ul |
| pSB1A3 | 2.5 ng/ul |
Digestion
| Reagent | Amount |
|---|---|
| 1-23L | 0.5 uL |
| 10x M buffer | 5 uL |
| 0.1%BSA | 5 uL |
| Xba I | 4 uL |
| Pst I | 0.5 uL |
| DW | 35 uL |
| Total | 50 uL |
| Reagent | Amount |
|---|---|
| 1-14K | 1.5 uL |
| 10x H buffer | 2 uL |
| 0.1% BSA | 2 uL |
| EcoR I | 1 uL |
| Spe I | 0.5 uL |
| DW | 13 uL |
| Total | 20 uL |
| Reagent | Amount |
|---|---|
| pSB1A3 | 20 uL |
| 10x H buffer | 3 uL |
| 0.1% BSA | 3 uL |
| EcoR I | 0.5 uL |
| Pst I | 0.5 uL |
| DW | 3 uL |
| Total | 30 uL |
| Reagent | Amount |
|---|---|
| pSB1A3 | 30 uL |
| 10x H buffer | 5 uL |
| 0.1% BSA | 5 uL |
| EcoR I | 0.5 uL |
| Pst I | 0.5 uL |
| DW | 9 uL |
| Total | 30 uL |
- Incubated each solution at 37C
- Solution of 1-23L was incubated for 150 min
- Solution of 1-14K was incubated for 90 min
- 30ul of pSB1A3 solution was incubated for 60 min
- 50ul of pSB1A3 solution was incubated for 30 min
- Performed electrophoresis for each solution
- put 12uls each into wells of a gel like below.
| Lane | DNA |
| 1 | λ/HindIII, EcoR I |
| 2~3 | 1-14K |
| 4~8 | 1-23L |
| 9~16 | pSB1A3 |
Results
- Could not see bands of 1-23L because leaked out
- Drove current for too long
- Extracted the other samples from a gel using promega kit
- Stored all at -20C.
