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Team:HokkaidoU Japan/Notebook/September16
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== Digestion of GFP and Double Terminator == | == Digestion of GFP and Double Terminator == | ||
| - | ==Parts Information== | + | ===Parts Information=== |
{| class="protocol" | {| class="protocol" | ||
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| - | + | * 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. | |
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| + | {|style="text-align:center;" class="protocol" | ||
| + | |- | ||
| + | |1-14K | ||
| + | |200 ng/ul | ||
| + | |- | ||
| + | |1-23L | ||
| + | |120 ng/ul | ||
| + | |- | ||
| + | |pSB1A3 | ||
| + | |2.5 ng/ul | ||
| + | |} | ||
===Digestion Menu=== | ===Digestion Menu=== | ||
Revision as of 07:41, 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 37℃ 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 6×sample buffer.put 12uls each into wells of a gel.
| 1 | λ/HindIII, EcoR I |
| 2~3 | 1-14K |
| 4~8 | 1-23L |
| 9~16 | pSB1A3 |
- cannot see 1-23L because of overflowing.extracted the other samples from a gel.dissolve them with 50ul of Nuclease free water.And they were stocked to freeze in -20℃.
