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

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    <h1>Material and Method</h1>
 
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    <h2>Assemble of DNA Nano-Cube</h2>
 
  
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    <h3>1. Thermal Cycler</h3>
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= Material and Method =
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    <p>
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== Assemble of DNA Nano-cube ==
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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. The temperature was then maintained for 5 minutes.
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  <!-- Polymerase Chain Reaction (PCR) -->
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    <p>
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Then the temperature will be lowered 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.
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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.
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    </p>
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    <h3>2. Native Polyacrylamide gel electrophoresis <small>(By HKU)</small></h3>
 
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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.
 
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[[File:Material_and_Method_1.jpg |thumbnail| center | 500px| Fig 4. Annealing process of the nano-cube. The above figure shows that the whole annealing process involves eight oligos. The eight oligos will form a nano-cube when the temperature is lowered from 95°C to room temperature.]]
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      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.
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    </p>
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    <h2>Peroxidase assay</h2>
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== Native Polyacrylamide gel electrophoresis (Collaborated with HKU Team) ==
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    <p>
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      To proof the presence of the formed nano-cube, we adopted 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic 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 H2O2 (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.
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    <h2>Cloning</h2>
 
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      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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Polyacrylamide gel electrophoresis (PAGE) is conducted to check whether the oligos are assembled as we designed. With assistance from iGEM HKU, 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.
  
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For the analysis of target binding, equimolar (10μM final) DNA nanostructure and nucleic acid input are mixed and incubated 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.
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  <h1>Experiments</h1>
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== Peroxidase assay ==
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  <p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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To prove the presence of the formed nano-cube, we conducted peroxidase assay by making use of a peroxidase activity of G-quadruplex. The target is RNA instead of DNA. However, to prove the concept, DNA peroxidase assay was done first because of its stability. RNA peroxidase assay was then followed.
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  Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.  
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In our assay, nanocube (5 nM final), and hemin (10μM) are added to buffer. The ABTS and Hemin were purchased from Tin Hang Technology Ltd. The mixture was incubated at room temperature for 15 minutes in a <I>Dlab rocking shaker</I> at 60rpm. 15μL ABTS solution and H<sub>2</sub>O<sub>2</sub> (20mM final) are added to the mixture. The reaction mixture was then transferred to microplate and was read by <I>Dymanica Halo LED 96-well Microplate Reader</I> with an absorbance of 405 nm.
  
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  <h5>What should this page contain?</h5>
 
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  <ul>
 
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  <li> Protocols </li>
 
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  <li> Experiments </li>
 
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  <li> Documentation of the development of your project </li>
 
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  <h5>Inspiration</h5>
 
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  <ul>
 
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  <li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
 
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  <li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
 
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  <li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
 
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Latest revision as of 07:15, 13 December 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. The temperature was then maintained for 5 minutes.

Then the temperature will be lowered 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.


Fig 4. Annealing process of the nano-cube. The above figure shows that the whole annealing process involves eight oligos. The eight oligos will form a nano-cube when the temperature is lowered from 95°C to room temperature.

Native Polyacrylamide gel electrophoresis (Collaborated with HKU Team)

Polyacrylamide gel electrophoresis (PAGE) is conducted to check whether the oligos are assembled as we designed. With assistance from iGEM HKU, 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 incubated 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

To prove the presence of the formed nano-cube, we conducted peroxidase assay by making use of a peroxidase activity of G-quadruplex. The target is RNA instead of DNA. However, to prove the concept, DNA peroxidase assay was done first because of its stability. RNA peroxidase assay was then followed.

In our assay, nanocube (5 nM final), and hemin (10μM) are added to buffer. The ABTS and Hemin were purchased from Tin Hang Technology Ltd. The mixture was incubated at room temperature for 15 minutes in a Dlab rocking shaker at 60rpm. 15μL ABTS solution and H2O2 (20mM final) are added to the mixture. The reaction mixture was then transferred to microplate and was read by Dymanica Halo LED 96-well Microplate Reader with an absorbance of 405 nm.