Notebook

Protocols used in this study

1. Basic Protocols
   • Plasmid Isolation Using Alkaline Lysis Method
     1. Inoculate transformed cell into LB medium supplemented with appropriate antibiotics
     2. Incubate culture at 37˚C, 200 rpm for overnight
     3. Discard supernatan, and repeat the step until all culture is used
     4. Add 100 µl Alkaline Lysis Solution I (0.05 M glucose; 0.025 M Tris-Cl pH 8.0; 0.01 EDTA pH 8.0) and resuspend cell pellet completely
     5. Add 200 µl Alkaline Lysis Solution II (0.2 N NaOH; 1% SDS), and invert tube several times. The solution should turn into transparent white, indicating the cell is lysed
     6. Add 150 µl Alkaline Lysis Solution III (3M Pottasium Acetate pH 7.2; 11.5% Glacial Acetate), and invert tube several times. The chromosomal DNA should make white floating thread
     7. Incubate on ice for 3-5 minutes
     8. Centrifuge at full speed for 5 minutes. At this point, plasmid DNA is dissolved in the supernatan while chromosomal DNA turn into white precipitate on the bottom of the tube
     9. Transfer supernatan to a new 1.5 microtube, add 2X volumes of isopropanol/absolute ethanol to precipitate plasmid DNA
     10. Centrifuge at full speed for 5 minutes, and discard supernatan
     11. Add 500 µl 70% ethanol to precipitated DNA
     12. Centrifuge at full speed for 5 minutes, and discard supernatan
     13. Let the precipitated DNA dreid by putting the microtube upside-down on a piece of tissue paper
     14. Resuspend plasmid by adding 50 µl TE buffer/Nuclease Free Water
   • Polymerase Chain Reaction for Restriction-Free Cloning
     1. Mix the following component in a PCR tube, for step I PCR