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
     
     2. Quick-spin the PCR tube, then place on the thermal cycler
     3. Set the PCR cycle profile as follow and then run as follows
     
     4. Confirm the amplicon by running the product on 1% agarose gel, 70V for 35 minutes
     5. If positive, continue to purified PCR product using GenepHlow Gel/PCR Purification Kit (Promega)
     6. Mix the following component in a PCR tube for step II PCR
     
     7. Quick spin the PCR tube, the place on the thermal cycler
     8. Set the PCR cycle profile as follow and the run
     
     9. Confirm the amplicon by running the PCR product on 1% agarose gel, 70V for 35 minutes
   • Restriction
     1. Mix the following mixture in a PCR tube
     
     2. Quick spin the PCR tube, then incubate at 37˚C for 30 minutes-60 minutes
     3. Incubate at 80˚C for 10 minutes to deactivate enzyme
     4. Confirm product by running on 1% agarose gel, 70V for 35 minutes
   • Purification using GenepHlow Gel/PCR Purification Kit (Promega)
   • Chemically competent E. coli cell
     1. Inoculate 5 ml glycerol stock of E. coli to 10 ml of LB medium
     2. Incubate culture at 37˚C, 200 rpm for overnight
     3. Transfer 1% of the overnight-grown cell into a new LB medium
     4. Incubate culture at 37˚C, 200 rpm until it reaches OD 0.3-0.4
     5. Aliquote culture into 1.5 ml microtube
     6. Centrifuge at 3.0000 xg, 4˚C for 10 minutes, discard the supernatant
     7. Add 480 µl cold CCMB buffer, and resuspend the cell pellet
     8. Incubate on ice for 20 minutes
     9. Centrifuge at 3.0000 xg, 4˚C for 10 minutes, discard the supernatant
     10. Add 60 µl cold CCMB buffer, and resuspend the cell pellet
     11. Incubate on ice for 20 minutes
     12. Store at -80˚C until use
   • Heat-shock transformation
     1. Incubate competent E. coli cell on ice for 10 minutes
     2. Add 5-10 µl plasmid into 60 µl of competent E.coli cell, place on ice for 30 minutes
     3. Incubate on 42˚C for 90 seconds for heat shock treatment
     4. Place back on ice and incubate for 2 minutes
     5. Add 600 µl of LB medium and resuspend
     6. Incubate culture at 37˚C, 200 rpm for 3 hours
     7. Centrifuge at 11.ooo xg for 1 minutes, and discard 550 µl of the supernatant
     8. Resuspend the cell pellet with the remaining supernatan
     9. Inoculate 50 µl of transformed E. coli cell on LB agar medium supplemented with appropriate antibiotics using spread technique
     10. Incubate at 37˚C overnight
2. Specialized Protocols