Team:SUSTC-Shenzhen/Notebook/CRISPR/Enzyme-digestion-agrose-gel-DNA-Extraction-for-PX330-vector

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Team SUSTC-Shenzhen

Notebook

Elements of the endeavor.

Contents


Enzyme digestion & agrose gel DNA Extraction for PX330 vector

2014/8/9 To prepare PX330 vector for the following ligation experiments

Materials

  • Enzyme & Buffer (New England Biolabs): EcoRI, EcoRVH, AgeI,
  • 10X cutsmart buffer
  • TIANgel Purification Kit

Methods

  1. Restriction enzyme digestion for plasmid PX330:

    Digestion system：(unit: ul)

    Total volume 10*buffer DNA EcoRI EcoRVHF AgeI ddH2O
    PX330 20 2 8 1 1 1 7
    PX330 20 2 8 1 1 1 7

    [According to the manual, 1ug DNA ~ 1 ul EcoRI/EcoRVHF/AgeI] Time: 2014.8.10 13:16 ~ 2014.8.10 18:44(digestion time: 5.5h)

  2. Electrophoresis for digestion system & Agrose gel DNA Extraction
    1. Electrophoresis for digestion system Loading system for digestion system:(unit: ul)
      Total volume / well DNA Dye TAE
      48 20 8 20

      Running conditions：110V, 18:44-19:30(45min)

    2. Agrose gel DNA Extraction [to remove enzymes (EcoRI ect.) & NEBuffer] Performed as the protocol provided by TIANgel Purification Kit, except that we eluted the DNA sample twice at the last step.

Results

Concentration 260/280
PX330 skeleton 14.0 8.40


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