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Team:HokkaidoU Japan/Notebook/September17
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| - | {{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September15|September 15]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/ | + | {{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September15|September 15]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September20|September 20]]</div></div> |
*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 | ||
| Line 16: | Line 16: | ||
<!-- Description -->|GFP | <!-- Description -->|GFP | ||
<!-- BioBrick No. -->|BBa_E0040 | <!-- BioBrick No. -->|BBa_E0040 | ||
| - | <!-- Well No. -->|[[Team:HokkaidoU_Japan/ | + | <!-- Well No. -->|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]] |
<!-- Length -->|720bp | <!-- Length -->|720bp | ||
<!-- Plasmid -->|pSB1A3 | <!-- Plasmid -->|pSB1A3 | ||
| Line 23: | Line 23: | ||
<!-- Description -->|double terminator | <!-- Description -->|double terminator | ||
<!-- BioBrick No. -->|BBa_B0015 | <!-- BioBrick No. -->|BBa_B0015 | ||
| - | <!-- Well No. -->|[[Team:HokkaidoU_Japan/ | + | <!-- Well No. -->|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] |
<!-- Length -->|129bp | <!-- Length -->|129bp | ||
<!-- Plasmid -->|pSB1AK3 | <!-- Plasmid -->|pSB1AK3 | ||
| Line 40: | Line 40: | ||
| - | * Performed electrophoresis of [[Team:HokkaidoU_Japan/ | + | * Performed electrophoresis of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]] and [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] to estimate concentration of each solution. |
* Estimated concentration from photo of electrophoresis | * Estimated concentration from photo of electrophoresis | ||
* pSB1A3 solution was done by other person. | * pSB1A3 solution was done by other person. | ||
* Made digestion recipes(below) based on estimated concentrations | * Made digestion recipes(below) based on estimated concentrations | ||
| - | * Made | + | * Made 30 ul of pSB1A3 solution, but latter found it insufficient to ligate parts |
** made more 50ul of it after | ** made more 50ul of it after | ||
| Line 53: | Line 53: | ||
!Amount | !Amount | ||
|- | |- | ||
| - | |[[Team:HokkaidoU_Japan/ | + | |[[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]] |
|200 ng/ul | |200 ng/ul | ||
|- | |- | ||
| - | |[[Team:HokkaidoU_Japan/ | + | |[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] |
|120 ng/ul | |120 ng/ul | ||
|- | |- | ||
| Line 69: | Line 69: | ||
!Amount | !Amount | ||
|- | |- | ||
| - | |[[Team:HokkaidoU_Japan/ | + | |[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] |
|0.5 uL | |0.5 uL | ||
|- | |- | ||
| Line 96: | Line 96: | ||
!Amount | !Amount | ||
|- | |- | ||
| - | |[[Team:HokkaidoU_Japan/ | + | |[[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]] |
|1.5 uL | |1.5 uL | ||
|- | |- | ||
| Line 177: | Line 177: | ||
* Incubated each solution at 37C | * Incubated each solution at 37C | ||
| - | * Solution of [[Team:HokkaidoU_Japan/ | + | * Solution of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] was incubated for 150 min |
| - | * Solution of [[Team:HokkaidoU_Japan/ | + | * Solution of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]] was incubated for 90 min |
| - | * | + | * 30 ul of pSB1A3 solution was incubated for 60 min |
| - | * | + | * 50 ul of pSB1A3 solution was incubated for 30 min |
* Performed electrophoresis for each solution | * Performed electrophoresis for each solution | ||
| - | * put | + | * put 12 uls each into wells of a gel like below. |
{|class="protocol" | {|class="protocol" | ||
| Line 203: | Line 203: | ||
'''Results''' | '''Results''' | ||
| - | * Could not see bands of [[Team:HokkaidoU_Japan/ | + | * Could not see bands of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] because leaked out |
** Drove current for too long | ** Drove current for too long | ||
* Extracted the other samples from a gel using promega kit | * Extracted the other samples from a gel using promega kit | ||
* Stored all at -20C. | * Stored all at -20C. | ||
Latest revision as of 08:27, 27 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 30 ul 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
- 30 ul of pSB1A3 solution was incubated for 60 min
- 50 ul of pSB1A3 solution was incubated for 30 min
- Performed electrophoresis for each solution
- put 12 uls 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.
