Team:HokkaidoU Japan/Notebook/August18

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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 By comparison toLambda/Hind III (ng/10 uL) ng/uL ratio size (bp) (ng) required (uL) used
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 and Transformation

Reagent Amount
DNA solution 6.3 uL
Ligation solution 6.3 uL
T4 ligase 1 uL
Total 13.6 uL
  • Incubated at 16C for 30 min
  • Transformation: Added all to 50 uL of competent cell
  • Incubated at 0C for 30 min
  • Heat shocked at 42C for 60 sec
  • 5 min on ice
  • Added 100 uL of LB
  • Incubated at 37C for 120 min
  • Spread onto the LBC plate
  • ncubated at 37C for 15~20 hrs

RBS Retry

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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