DNA Techniques

  1. Plasmid Preparation

    1. Mini prep

      1. Alkaline lysis method

      2. Boiling method

    2. Large prep

      1. Large scale

      2. Sue’s large scale

      3. Differential precipitation

  2. Preparation of DNA from Cultured Cells

    1. High molecular weight

    2. Hirt method

    3. Total DNA extraction from mammalian cells

  3. DNA Electrophoresis

    1. Agarose – horizontal and vertical

    2. Polyacrylamide

    3. Alkaline agarose

  4. Cloning Techniques

    1. Electroelution

    2. Acrylamide crush and soak

    3. 1c. low-melt agarose

    4. Fragment isolation

      1. Ligation

      2. Quick ligation

  5.  Sequencing

    1. Dideoxy (Sanger)

      1. Dideoxy sequencing with 35S

      2. Sequencing double-stranded DNA

      3. Dideoxy working solutions

      4. Automated dideoxy sequencing

    2. Maxam-Gilbert

    3. Big-Dye sequencing

  6.  Modification

    1. Phosphatase treatment

      1. Calf intestinal phosphatase

      2. Bacterial alkaline phosphatase

    2. Kinase treatment

    3. Conversion of 5′ ends to blunt ends

    4. 3′ end filling

    5. Nick translation

    6. Restriction enzyme analysis

    7. Multiprime labeling

    8. Mutagenesis

  7. Amplification

    1. Polymerase chain reaction

    2. Quantitative RT-PCR

    3. Real-time PCR

    4. Colony PCR