Difference between revisions of "Team:HK SKHLPSS/Experiments"

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     <h1>Material and Method</h1>
 
     <h1>Material and Method</h1>
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     <h2>Assemble of DNA Nano-Cube</h2>
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     <h2>Assemble of DNA Nano-cube</h2>
  
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    <h3>Thermal Cycler</h3>
 
 
     <p>
 
     <p>
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       Eight DNA strands are mixed and placed in the thermal cycler. The thermal cycler is then raised the mixture’s temperature until 95°C for 5 minutes and then held for 5 minutes
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       Eight DNA strands are mixed and placed in the <I>Dynamica C-Master Gradient thermal cycler</I>. The thermal cycler raised the mixture’s temperature to 95°C for 5 minutes and then held for 5 minutes.
 
     </p>
 
     </p>
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  <!-- Polymerase Chain Reaction (PCR) -->
 
  
 
     <p>
 
     <p>
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      Then the temperature will be lower 1°C per minute from 95°C to room temperature to allow eight single strands to anneal in accordance with our design by Watson-Crick base pairing to form the nano-device.
+
    Then the temperature will be lower 1°C per minute from 95°C to room temperature to allow eight single strands to anneal in accordance with our design by Watson & Crick’s base pairing to form the nano-cube.
 
     </p>
 
     </p>
  
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     <h3>Native Polyacrylamide gel electrophoresis <small>(By HKU)</small></h3>
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     <h2>Native Polyacrylamide gel electrophoresis (Collaborated with HKU Team)</h2>
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  <!--  -->
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     <p>  
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     <p>
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       The assembly of DNA nanostructure is analyzed by 12% Polyacrylamide gel electrophoresis (PAGE) where the combinations of oligos (10μL, 200nM) are loaded. All the gels are run at a constant voltage of 100V. GelRed is used to stain the gels.
+
       Polyacrylamide gel electrophoresis (PAGE) is conducted to check whether the oligos are assembled as we designed. Collaborating with HKU team, the assembly of DNA nanostructure is analyzed by 12% PAGE where the combinations of oligos (10μL, 200nM) are loaded. All the gels are run at a constant voltage of 100V. GelRed is used to stain the gels.
 
     </p>
 
     </p>
  
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     <h2>Peroxidase assay</h2>
 
     <h2>Peroxidase assay</h2>
 +
 
     <p>
 
     <p>
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       To proof the presence of the formed nano-cube, we adopted 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) [ABTS] assay by making use of the peroxidase activity of G-quadruplex. In our assay, nano-cube (100 nM final), and hemin (77μM) are added to buffer. The mixture is incubated at room temperature for 15 minutes in a shaker (60rpm). 15μL ABTS solution and H<sub>2</sub>O<sub>2</sub> (20mM final) are added to the mixture. The reaction mixture is then transferred to a 96-well plate and absorbance at 405 nm is measured with a microplate spectrophotometer.
+
       For the analysis of target binding, equimolar (10μM final) DNA nanostructure and nucleic acid input are mixed and incubate at room temperature for 15 minutes in a shaker. The mixture (10μL, 200nM) is then loaded to 12% polyacrylamide gel. The PAGE is conducted at a constant voltage of 100V. GelRed is used to stain the gel.
 
     </p>
 
     </p>
  
−
    <h2>Cloning</h2>
 
−
    <p> 
 
−
      After success of in-vitro experiment, we would want to try this method in-vivo. The sticky ends produced then will anneal such that neither SpeI nor XbaI will recognize it. We can therefore fuse the G-block fragments by cutting one couple of G-block fragments one at a time with one RE applied in each, then ligate it to produce a ‘dimer’. Taking our p_O15 as an example, we will first digest the suffix of O1-fragment with SpeI and the prefix of O5-fragment with XbaI. Then we performed ligation. After that, we will digest the backbone and ligated O1-O8 fragment with PstI and EcoRI, and ligate them together to make it as a complete plasmid. The flowchart below illustrates our plan.
 
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    </p>
 
 
   </div>
 
   </div>
  

Revision as of 08:37, 27 October 2017

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Material and Method

Assemble of DNA Nano-cube

Eight DNA strands are mixed and placed in the Dynamica C-Master Gradient thermal cycler. The thermal cycler raised the mixture’s temperature to 95°C for 5 minutes and then held for 5 minutes.

Then the temperature will be lower 1°C per minute from 95°C to room temperature to allow eight single strands to anneal in accordance with our design by Watson & Crick’s base pairing to form the nano-cube.

Native Polyacrylamide gel electrophoresis (Collaborated with HKU Team)

Polyacrylamide gel electrophoresis (PAGE) is conducted to check whether the oligos are assembled as we designed. Collaborating with HKU team, the assembly of DNA nanostructure is analyzed by 12% PAGE where the combinations of oligos (10μL, 200nM) are loaded. All the gels are run at a constant voltage of 100V. GelRed is used to stain the gels.

For the analysis of target binding, equimolar (10μM final) DNA nanostructure and nucleic acid input are mixed and incubate at room temperature for 15 minutes in a shaker. The mixture (10μL, 200nM) is then loaded to 12% polyacrylamide gel. The PAGE is conducted at a constant voltage of 100V. GelRed is used to stain the gel.

Peroxidase assay

For the analysis of target binding, equimolar (10μM final) DNA nanostructure and nucleic acid input are mixed and incubate at room temperature for 15 minutes in a shaker. The mixture (10μL, 200nM) is then loaded to 12% polyacrylamide gel. The PAGE is conducted at a constant voltage of 100V. GelRed is used to stain the gel.