Team:HokkaidoU Japan/Notebook/August25

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Notebook

Transformation Results

Colony Check

Plate Colonies
M 34 no
M 12 yes
I 34 no
I 34 yes
1-3A 34 yes
1-3A 12 yes
  • 1-3A had colonies in lated on mediums containing 34 ug/uL of chloramphenicol
    • So the medium and antibiotic consentration seems ok,
  • Medium on which M 12 was plated was inoculated with chloramphenicol after solidification so there were some irregularities and colonies on the fringe
  • Colonies of 1-3A and 12 were obviously different

→ So we suspected issues in Ligation and DNA concentration bused for transformation

Estimation of 1-3A Consentration

Electrophoresis of 1uL of 1-3A DNA
  • Added 1 uL of 1-3A into 30 uL of competent cells
  • Transformation was successful
  • Electrophoresed 1 uL of 1-3A，calculated amount of transformed DNA

→Estimated concentration 2 ng/uL

pSB1C3をエタ沈で濃縮，Ligation

エタ沈

  1. 3 M 酢酸ナトリウム 8.2 uL加える
  2. 100%エタノールを205 uL加え，混ぜる
  3. 15,00rpm@4℃で10分間遠心する
  4. 上清を捨て，500 uL 70%エタノールを加え，混ぜる
  5. 15,00rpm@4℃で5分間遠心する
  6. 上清を捨て，真空デシケータで乾燥させる
  7. DW 8.2 uLを加え，元の10倍に濃縮する

Ligation

  1. 8.2 uL DNA solutionに同量のLigation Solutionを加える
  2. T4 ligase 1 uLを加え，16℃で30分間インキュベート
  3. 明日，電気泳動・ゲル抽・Transformation

らおりプライマーで作ったPCR productsのDigestion

HokkaidoU Japan 20100825c.JPG

前日のPCRから，増やすことのできたパーツのDNA量を求めた

RBS 27 ng/uL 312 bp
GFP 54 ng/uL 1020 bp
dT 49 ng/uL 429 bp

Digestion 反応系

Reagent Amount
10x M buffer 1 uL
DW 4.5
DNA 2.5
BSA 1
EcoR I 0.5
Pst I 0.5
Total 10 uL

→37℃で60分

→ラオリプライマーの真価を電気泳動で確認
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