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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/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 and Double Terminator ==
| |
| - | ===Parts Information===
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| - | {| class="protocol"
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| - | |-
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| - | |GFP
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| - | |BBa_E0040
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| - | |1-14K
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| - | |720bp
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| - | |pSB1A3
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| - | |-
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| - | |double terminator
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| - | |BBa_B0015
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| - | |1-23L
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| - | |129bp
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| - | |pSB1AK3
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| - | |-
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| - | |pSB1A3
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| - | |pSB1A3
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| - | | -
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| - | |2157bp
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| - | |pSB1A3
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| - | |}
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| - |
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| - | 1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.
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| - |
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| - |
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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.
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| - | * 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.
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| - | * 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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| - |
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| - | {|style="text-align:center;" class="protocol"
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| - | |-
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| - | |1-14K
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| - | |200 ng/ul
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| - | |-
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| - | |1-23L
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| - | |120 ng/ul
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| - | |-
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| - | |pSB1A3
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| - | |2.5 ng/ul
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| - | |}
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| - |
| |
| - | ===Digestion Menu===
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| - | {|style="text-align:center; float:left;" class="protocol"
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| - | |-
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| - | |1-23L
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| - | |0.5 uL
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| - | |-
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| - | |10x M buffer
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| - | |5 uL
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| - | |-
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| - | |0.1%BSA
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| - | |5 uL
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| - | |-
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| - | |Xba I
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| - | |4 uL
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| - | |-
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| - | |Pst I
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| - | |0.5 uL
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| - | |-
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| - | |DW
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| - | |35 uL
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| - | |-
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| - | |style="border-top:1px solid #000;"|'''Total'''
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| - | |style="border-top:1px solid #000;"|'''50 uL'''
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| - | |}
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| - |
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| - | {|style="text-align:center; float:left;" class="protocol"
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| - | |-
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| - | |1-14K
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| - | |1.5 uL
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| - | |-
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| - | |10x H buffer
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| - | |2 uL
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| - | |-
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| - | |0.1% BSA
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| - | |2 uL
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| - | |-
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| - | |EcoR I
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| - | |1 uL
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| - | |-
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| - | |Spe I
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| - | |0.5 uL
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| - | |-
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| - | |DW
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| - | |13 uL
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| - | |-
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| - | |style="border-top:1px solid #000;"|'''Total'''
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| - | |style="border-top:1px solid #000;"|'''20 uL'''
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| - | |}
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| - |
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| - | {|style="text-align:center; float:left;" class="protocol"
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| - | |-
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| - | |pSB1A3
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| - | |20 uL
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| - | |-
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| - | |10x H buffer
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| - | |3 uL
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| - | |-
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| - | |0.1% BSA
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| - | |3 uL
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| - | |-
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| - | |EcoR I
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| - | |0.5 uL
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| - | |-
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| - | |Pst I
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| - | |0.5 uL
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| - | |-
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| - | |DW
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| - | |3 uL
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| - | |-
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| - | |style="border-top:1px solid #000;"|'''Total'''
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| - | |style="border-top:1px solid #000;"|'''30 uL'''
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| - | |}
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| - |
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| - | {|style="text-align:center; float:left;" class="protocol"
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| - | |-
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| - | |pSB1A3
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| - | |30 uL
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| - | |-
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| - | |10x H buffer
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| - | |5 uL
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| - | |-
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| - | |0.1% BSA
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| - | |5 uL
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| - | |-
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| - | |EcoR I
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| - | |0.5 uL
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| - | |-
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| - | |Pst I
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| - | |0.5 uL
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| - | |-
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| - | |DW
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| - | |9 uL
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| - | |-
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| - | |style="border-top:1px solid #000;"|'''Total'''
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| - | |style="border-top:1px solid #000;"|'''30 uL'''
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| - | |}
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| - |
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| - | <div style="clear:both"></div>
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| - |
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| - | * put each solutions into 37C incubator.
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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.
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| - | * electrophoresed each solutions added 6x sample buffer.
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| - | * put 12uls each into wells of a gel like below.
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| - |
| |
| - | {|class="protocol"
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| - | |-
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| - | |1
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| - | |λ/''Hin''dIII, EcoR I
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| - | |-
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| - | |2~3
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| - | |1-14K
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| - | |-
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| - | |4~8
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| - | |1-23L
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| - | |-
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| - | |9~16
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| - | |pSB1A3
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| - | |}
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| - |
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| - | '''Result'''
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| - | * cannot see 1-23L because of overflowing.
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| - | * extracted the other samples from a gel.
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| - | * dissolve them with 50 ul of Nuclease free water, and they were stocked to freeze in -20C.
| |
"
