Team:HokkaidoU Japan/Notebook/August18

From 2010.igem.org

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===Ligationに必要なDNA Solutionの調製===
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===Preparation of DNA Solution for Ligation===
{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
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!パーツ
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!Part
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!λ/''Hin''d IIIと比べて (ng/10 uL)
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![https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png Lambda/''Hin''d III] と比べて (ng/10 uL)
!ng/uL
!ng/uL
!割合
!割合

Revision as of 16:26, 21 September 2010

Notebook

RBS digestion: Revenge

HokkaidoU Japan 20100818a.JPG
Reagent Amount
1-2M 10 uL
DW 4 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

→Incubated at 37C for 60 min

  • Added 4 uL of 6x Sample Buffer making it total of 12 uL per lane and electrophoresed

→Extracted for gel
→Electrophoresed 10 uL of Extracted DNA

Failure

  • After better inspection of Kit specs it came to lite that retreavel rate for 50 bp and less is 26%
    • RBS is just quite small when cut

Ligation

Preparation of DNA Solution for Ligation

Part Lambda/Hind III と比べて (ng/10 uL) ng/uL 割合 size (bp) 必要 (ng) 使用 (uL)
Vector 50 ng/10 uL 5 ng/uL 1 2996 bp 10 ng 2 uL
RFP 250 ng/10 uL 25 ng/uL 2 700 bp 7 ng 0.3 uL
double terminator 5 ng/10 uL 0.5 ng/uL 2 200 bp 2 ng 4 uL
Total 6.3 uL

Ligation & Transformation

Reagent Amount
DNA solution 6.3 uL
Ligation solution 6.3 uL
T4 ligase 1 uL
Total 13.6 uL
  • 16℃，30 min
  • competent cell 50 uLに全量加えた（Transformation）
  • 0℃，30 min
  • 42℃，60 sec
  • 5 min on ice
  • add LB 100 uL
  • 37℃，120 min
  • spread onto the LBC
  • 37℃，15~20 hrs

RBS 再リベンジ

Digestion

Reagent Amount
（RBS）1-2M 10 uL
DW 4 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

→37℃，60 min

エタ沈

  • 2 uLの3 M 酢酸ナトリウムを加えた
  • 44 uLのエタノールを加えた
  • 液体窒素の中で凍らせた（1.5 mLのチューブに移す）
  • 溶かしてから15,996 rpm @4℃で５分間遠心した
  • 上清をほかのチューブに移した（一応これも遠心し，上清を捨て，同じ操作をした）
  • 100 uLの70%エタノールで壁をリンスし，15,996 rpm @4℃で５分間遠心した
  • 上清を捨て，真空デシケータで乾燥させた
  • TE 5 uLで溶かした

電気泳動

HokkaidoU Japan 20100818b.JPG
  • TEで溶かしたDNA Solution 1 uLに6x SB 1 uLを加えた
  • 最初にとった上清も同じ操作をした
Lane DNA
2 TSUDA Marker 1
3 上清
4 DNA solution
  • DNA solutionにしっかり回収されていた
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