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Team:HokkaidoU Japan/Notebook/September16
From 2010.igem.org
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* 1-23L solution was put about a half and two hours, 1-14K solution was done about a half and an hour, 30ul of pSB1A3 solution was done about an hour, and 50ul of pSB1A3 was done about a half hour. | * 1-23L solution was put about a half and two hours, 1-14K solution was done about a half and an hour, 30ul of pSB1A3 solution was done about an hour, and 50ul of pSB1A3 was done about a half hour. | ||
* electrophoresed each solutions added 6x sample buffer. | * electrophoresed each solutions added 6x sample buffer. | ||
| - | * put 12uls each into wells of a gel | + | * put 12uls each into wells of a gel like below. |
| - | {| | + | {|class="protocol" |
|- | |- | ||
|1 | |1 | ||
Revision as of 07:46, 19 September 2010
since 13 Jul 2010
since 1 Aug 2010
Digestion of GFP and Double Terminator
Parts Information
| GFP | BBa_E0040 | 1-14K | 720bp | pSB1A3 |
| double terminator | BBa_B0015 | 1-23L | 129bp | pSB1AK3 |
| pSB1A3 | pSB1A3 | - | 2157bp | pSB1A3 |
1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.
- electrophoresed 1ul of 1-14K and 1-23L added 0.5ul of 6×sample buffer to estimate concentration of each solution.
- estimated concentration from photo of electrophoresys. But I forgot to electrophorese pSB1A3 solution with the other samples, so pSB1A3 solution was done by other person.
- made digestion recipes based on each concentrations(below). Why pSB1A3 recipe is two, because I firstly made 30ul of pSB1A3 solution, but I found it was insufficient to ligate parts, so I made more 50ul of it after.
| 1-14K | 200 ng/ul |
| 1-23L | 120 ng/ul |
| pSB1A3 | 2.5 ng/ul |
Digestion Menu
| 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 |
| 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 |
| 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 |
| 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 |
- put each solutions into 37C incubator.
- 1-23L solution was put about a half and two hours, 1-14K solution was done about a half and an hour, 30ul of pSB1A3 solution was done about an hour, and 50ul of pSB1A3 was done about a half hour.
- electrophoresed each solutions added 6x sample buffer.
- put 12uls each into wells of a gel like below.
| 1 | λ/HindIII, EcoR I |
| 2~3 | 1-14K |
| 4~8 | 1-23L |
| 9~16 | pSB1A3 |
Result
- cannot see 1-23L because of overflowing.
- extracted the other samples from a gel.
- dissolve them with 50 ul of Nuclease free water, and they were stocked to freeze in -20C.
