Difference between revisions of "Team:ITB Indonesia/Notebook"

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               <li>Incubate on ice for 3-5 minutes</li>
 
               <li>Incubate on ice for 3-5 minutes</li>
 
               <li>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</li>
 
               <li>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</li>
−
               <li>10. Transfer supernatan to a new 1.5 microtube, add 2X volumes of isopropanol/absolute ethanol to precipitate plasmid DNA</li>
+
               <li>Transfer supernatan to a new 1.5 microtube, add 2X volumes of isopropanol/absolute ethanol to precipitate plasmid DNA</li>
−
               <li>11. Centrifuge at full speed for 5 minutes, and discard supernatan</li>
+
               <li>Centrifuge at full speed for 5 minutes, and discard supernatan</li>
−
               <li>12. Add 500 µl 70% ethanol to precipitated DNA</li>
+
               <li>Add 500 µl 70% ethanol to precipitated DNA</li>
−
               <li>13. Centrifuge at full speed for 5 minutes, and discard supernatan</li>
+
               <li>Centrifuge at full speed for 5 minutes, and discard supernatan</li>
−
               <li>14. Let the precipitated DNA dreid by putting the microtube upside-down on a piece of tissue paper</li>
+
               <li>Let the precipitated DNA dreid by putting the microtube upside-down on a piece of tissue paper</li>
−
               <li>15. Resuspend plasmid by adding 50 µl TE buffer/Nuclease Free Water</li></ol>
+
               <li>Resuspend plasmid by adding 50 µl TE buffer/Nuclease Free Water</li></ol>
 
         </li>
 
         </li>
−
         <li>Tea</li>
+
         <li>Polymerase Chain Reaction for Restriction-Free Cloning
 +
                    <ol>
 +
                        <li>Mix the following component in a PCR tube, for step I PCR</li>
 +
                        <
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
</li>
 
         <li>Milk</li>
 
         <li>Milk</li>
 
</ul>
 
</ul>

Revision as of 01:49, 2 November 2017

Team Attributions
Collaborations Overview Design Notebook Contribution Results Improve
Basic Parts Composite Part Part Collection
Silver HP Integrated and Gold Public Engagement


Notebook


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. <
      2. Milk
  2. Specialized Protocols