|
|
| (8 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| | {{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/September17|September 17]]</div></div> | | {{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/September17|September 17]]</div></div> |
| - |
| |
| - | *digestion of GFP(1-14K) and double terminator(1-23L)
| |
| - |
| |
| - |
| |
| - | == Digestion of GFP and Double Terminator ==
| |
| - | ==Parts Information==
| |
| - | {| class="protocol"
| |
| - | |-
| |
| - | |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.
| |
| - |
| |
| - |
| |
| - | ==Method==
| |
| - |
| |
| - | # electrophoresed 1μl of 1-14K and 1-23L added 0.5μl 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.Why pSB1A3 recipe is two,because I firstly made 30μl of pSB1A3 solution,but I found it was insufficient to ligate parts,so I made more 50μl of it after.
| |
| - |
| |
| - | ==Estimates of concentration==
| |
| - |
| |
| - | 1-14K:200ng/μl
| |
| - |
| |
| - | 1-23L:120ng/μl
| |
| - |
| |
| - | pSB1A3:2.5ng/μl
| |
| - |
| |
| - |
| |
| - | ===Digestion Menu===
| |
| - | {|style="text-align:cente; float:left;" class="protocol"
| |
| - | |-
| |
| - | |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
| |
| - | |-
| |
| - | |style="border-top:1px solid #000;"|'''Total'''
| |
| - | |style="border-top:1px solid #000;"|'''50 uL'''
| |
| - | |}
| |
| - |
| |
| - | {|style="text-align:cente; float:left;" class="protocol"
| |
| - | |-
| |
| - | |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
| |
| - | |-
| |
| - | |style="border-top:1px solid #000;"|''Total''
| |
| - | |style="border-top:1px solid #000;"|''20 uL''
| |
| - | |}
| |
| - | {|style="text-align:cente; float:left;" class="protocol"
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |style="border-top:1px solid #000;"|
| |
| - | |style="border-top:1px solid #000;"|
| |
| - | |}
| |
| - | {|style="text-align:cente; float:left;" class="protocol"
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |
| |
| - | |
| |
| - | |-
| |
| - | |style="border-top:1px solid #000;"|
| |
| - | |style="border-top:1px solid #000;"|
| |
| - | |}
| |
| - | 1.5μl
| |
| - |
| |
| - | H-buffer2 μl
| |
| - |
| |
| - | 0.1%BSA2 μl
| |
| - |
| |
| - | Eco1 μl
| |
| - |
| |
| - | Spe0.5μl
| |
| - |
| |
| - | DW13μl
| |
| - |
| |
| - |
| |
| - | pSB1A320μl
| |
| - |
| |
| - | H-buffer3μl
| |
| - |
| |
| - | 0.1%BSA3 μl
| |
| - |
| |
| - | Eco0.5 μl
| |
| - |
| |
| - | Pst0.5μl
| |
| - |
| |
| - | DW3μl
| |
| - |
| |
| - |
| |
| - | pSB1A330μl
| |
| - |
| |
| - | H-buffer5μl
| |
| - |
| |
| - | 0.1%BSA5μl
| |
| - |
| |
| - | Eco0.5 μl
| |
| - |
| |
| - | Pst0.5μl
| |
| - |
| |
| - | DW9μl
| |
| - |
| |
| - |
| |
| - | 4)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,30μl of pSB1A3 solution was done about an hour,and 50μl of pSB1A3 was done about a half hour.
| |
| - |
| |
| - |
| |
| - | 5)electrophoresed each solutions added 6×sample buffer.put 12μls each into wells of a gel.
| |
| - |
| |
| - | 1:λ/HindⅢ EcoRⅠ
| |
| - |
| |
| - | 2~3:1-14K
| |
| - |
| |
| - | 4~8:1-23L
| |
| - |
| |
| - | 9~16:pSB1A3
| |
| - |
| |
| - |
| |
| - | 6)cannot see 1-23L because of overflowing.extracted the other samples from a gel.dissolve them with 50μl of Nuclease free water.And they were stocked to freeze in -20℃.
| |
"
