IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-20

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Contents

double digesting E0040 and I7100 for ligation

  • Digesting E0040 and I7100 with EcoR1/SpeI.
  • Digestion system contains (totally 19uL): 2uL 10*H, 0.5uL EcoRI, 0.5uL PstI, 6uL plasmid, 10uL ddH2O.
  • 37℃ culutre for 12 hours.

electrophoresis result

  • from left to right:
    • 1 I7100 plasmid
    • 2-5 I7100 and EcoR1 and Pst1
    • 6 E0040 plasmid
    • 7-10 E0040 and EcoR1 and Pst1
    • 11 marker DL200 plus

Image:2007-7-20_I7100_EcoR1+Pst1_and_E0040_EcoR1+Pst1.jpg‎

Gel Purification

  • extract pSB3k3(I7100) vector and e0040 fragments from gel

electrophoresis test the products of gel extraction

  • Digestion system contains:2uL plasmid,2uL Loading Buffer,2uL Dye.
  • from left to right:
  1. EGFP(E0040)
  2. pSB3k3(I7100)
  3. Marker DL200 plus

Image:2007-7-20_EGFP(E0040)_and_pSB3k3(I7100).jpg

ligation

  • ligate the pSB3k3(I7100) and EGFP(E0040) with T4 ligase

mini-prep I7100 and E0040

single digestion test

  • Digesting E0040 and r0040 with EcoR1.
  • Digestion system contains (totally 20uL): 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O.
  • 37℃ culutre for 3 hours.

electrophoresis result

  • from left to right:
  1. 1-3 e0040 and EcoR1
  2. 4-5 r0040 and EcoR1
  3. 6 Marker

Image:E0040-3-4-5_EcoR1 and R0040-2-4_EcoR1.jpg

  • ! there are abnormal bands in the e0040 samples.

another single digestion test

  • digest I7100 with Xba1
  • digest e0040(another sample) with EcoR1
  • Digestion system contains (totally 20uL):
  1. I7100: 2uL 10*M, 0.5uL XbaI, 4uL plasmid, 13.5uL dH2O.
  2. E0040: 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O.

electrophoresis result

  • From left to right:
  1. 1 I7100 plasmid
  2. 2-4 I7100 and Xba1
  3. 5 e0040 plasmid
  4. 6-8 e0040 and EcoR1
  5. 9 marker DL2000 plus
  6. 10 r0040 and EcoR1 (products of former digestion test)

Image:I7100-3-4-5 XbaI and E0040-6-7-8 EcoRI and R0040-4 EcoRI.jpg

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