Team:HokkaidoU Japan/Notebook/August13

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=ミニプレで増えなかったパーツのPCR=
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=PCR of parts which didn't amplify well via mini prep=
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==反応液の組成==
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==Composition of Reaction Solution==
{|class="protocol"
{|class="protocol"
|-
|-
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|style="text-align:left;"| 1 uL
|style="text-align:left;"| 1 uL
|-
|-
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|style="text-align:right;"| Template (1-18F) :  
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|style="text-align:right;"| Template ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]]) :  
|style="text-align:left;"| 1 uL
|style="text-align:left;"| 1 uL
|-
|-
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|style="text-align:right; border-top:1px solid #000;"|'''Total''' :  
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|style="text-align:right; border-top:1px solid #996;"|'''Total''' :  
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|style="text-align:left; border-top:1px solid #000;"| 50 uL
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|style="text-align:left; border-top:1px solid #996;"| 50 uL
|}
|}
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==primerの除去==
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==Removal of Primers==
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# PCR products 50 uLにTE 150 uLを加え，ろ過用のチューブ（Microcon YM-10）に入れ，14000Gで20分間遠心した．
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# Added 150 uL of TE to 50 uL of PCR product, each
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# 十分量ろ過されていなかったので，もう10分遠心した
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# Transfered into Microcon YM-10 filter and cetrifuged for 20 min at 14,000 G
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# ろ液を測ると140 uLだっため，さらに10分遠心した
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# Much of the solution remained so centrifuged for aditional 10 min
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# なんかまだびみょうだったからもう10分遠心した
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# Measured the amount left
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# DNAを含む溶液が19 uLになったため，31 uLのTEを加え総量50 uLにした
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## It was 140 uL so centrifuged again for 10min
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# And again
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# Finally DNA solution was reduced to 19 uL so we added 31 uL to make final volume of 50 uL
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==電気泳動==
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==Electrophoresis==
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[[Image:HokkaidoU Japan 20100813a.jpg‎|200px|right|thumb|]]
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# primerなどを除去したDNA溶液 1 uLに6x Sample Buffer 0.4 uLを加え，泳動した
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[[Image:HokkaidoU Japan 20100813a.jpg‎|200px|right|thumb|PCR for DNA confirmation]]
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# 同時に，ろ液 10 uLに6x Sample Buffer 2 uLを加えたものも遠心した
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* マーカーはpUC119/''Hin''f
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# Added 0.4 uL of 6x Sample Buffer to 1 uL of DNA solution and electrophoresed it.
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* PCRでDNAが増幅されたことが確認された(543 bpと1330 bpの間に約700 bpのPCR産物)
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# At the same time add added 2 uL of 6x Sample Buffer to 10 uL of flow trough and centrifuged
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* 油断して45分間泳動したため，同時に流したprimerを含むろ液は流れた
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* Used marker [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC119/''Hin''f]
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* It was confirmed that DNA was amplified by PCR
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** Between marker bands of 543 bp and 1330 bp PCR product band of 700 bp was visible
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* Due to negligence (electrophoresis for 45 min), flow through with primers flowed through and exited gel
<div style="clear:both">
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Latest revision as of 07:26, 27 October 2010

Notebook

PCR of parts which didn't amplify well via mini prep

Composition of Reaction Solution

Reagent Amount
Autoclaved DW : 33 uL
10x PCR buffer : 5 uL
2 mM dNTPs : 5 uL
25 mM MgSO4 : 3 uL
EX-F primer : 1 uL
PS-R primer : 1 uL
KOD plus Neo : 1 uL
Template (1-18F) : 1 uL
Total : 50 uL

Removal of Primers

  1. Added 150 uL of TE to 50 uL of PCR product, each
  2. Transfered into Microcon YM-10 filter and cetrifuged for 20 min at 14,000 G
  3. Much of the solution remained so centrifuged for aditional 10 min
  4. Measured the amount left
    1. It was 140 uL so centrifuged again for 10min
  5. And again
  6. Finally DNA solution was reduced to 19 uL so we added 31 uL to make final volume of 50 uL

Electrophoresis

PCR for DNA confirmation
  1. Added 0.4 uL of 6x Sample Buffer to 1 uL of DNA solution and electrophoresed it.
  2. At the same time add added 2 uL of 6x Sample Buffer to 10 uL of flow trough and centrifuged
  • Used marker pUC119/Hinf
  • It was confirmed that DNA was amplified by PCR
    • Between marker bands of 543 bp and 1330 bp PCR product band of 700 bp was visible
  • Due to negligence (electrophoresis for 45 min), flow through with primers flowed through and exited gel
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