Team:HokkaidoU Japan/Notebook/September16

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(Digestion Menu)
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== Digestion of GFP and Double Terminator ==
 
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===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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1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.
 
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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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{|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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{|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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{|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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{|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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<div style="clear:both"></div>
 
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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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'''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.
 

Latest revision as of 17:35, 19 September 2010