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Latest revision as of 07:24, 29 August 2014

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<ul> 

  	    <li class='active '><a href="http://openwetware.org/wiki/Biomod/2014/Tianjin"><span>Home</span></a></li>
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  	            <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#background'><span>Background</span></a></li>
                   <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#motivation'><span>Motivation</span></a></li>
                   <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#calculation'><span>Calculation</span></a></li>
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  	            <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#strand replacement reaction"><span>Strand Replacement Reaction</span></a></li>
                   <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#synthesis of au-dna-cy3"><span> Synthesis of AU-DNA-CY3</span></a></li>
                   <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#dna origami"><span>DNA Origami</span></a></li>
               </ul> 			
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           <li class='active'><a href="http://openwetware.org/wiki/Biomod/2014/result.html"><span>Protocol</span></a>

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CY3 NHS ester labelingt:

(1).Calculate required amount of NHS ester:

NHS ester weight [mg] = 8 × amino compound weight [mg] × NHS ester molar weight [Da] / amino compound molar weight [Da].

In our experiments, in order to label 100ug of amino-modified DNA (molar weight 10927 Dalton) with Cy3 NHS ester (molar weight 590.15Dalton), and obtain maximum yield of mono-labeled product, we use

8× 100ug × 590.15 Da /10297 Da = 45.85 ug of Cy3 dye NHS ester.

(2).Determine volume of reaction mixture: use 10ul volume which concentration is 5ug of amino-modified DNA per uL of mixture.

(3).Dissolve 1mg CY3 NHS ester in 22ul of DMF. After the reaction, NHS ester can be stored in solution for 1-2 months at -20ºC.

(4).Dissolve amino-modified DNA in 9ul of 0.1 M Tris buffer with pH 8.3-8.5. Note pH is the most important thing.

(5).Add 1ul CY3 NHS ester solution to the solution of DNA, and vortex well. Keep the mixture at room temperature during at least 4 hours.

(6).Purify the conjugate using ethanol precipitation.


Synthesis of AU-DNA-CY3:

(1).Prepare 10 mM TCEP

(2).Prepare 13 nM gold nanoparticles: two kinds of gold nanoparticles with diameter of 13nm and 40nm

(3).Pipette 9μl of 1 mM DNA into a microcentrifuge tube

(4).Add 1μl of 500 mM acetate buffer (pH 5.2) and 1.5μl of 10 mM TCEP to the tube to activate the thiol-modified DNA. Incubate the sample at room temperature for 1 h.

(5).Transfer 3 ml of the already prepared gold nanoparticles （13 nM）to the centrifuge tube, and then add the TCEP-treated thiol DNA with gentle shaking by hand.

(6).Cap the centrifuge tube and store them in a drawer at room temperature for at least 16 h.

(7).Add 30 μl of 500 mM Tris acetate (pH 8.2) buffer drop wise to each tube with gentle hand shaking. The final Tris acetate concentration is 5 mM.

(8).Add 300 μl of 1 M NaCl dropwise to each tube with gentle hand shaking. Store the tube in a drawer for at least another day before use.

(9).Transfer 500 μl of functionalized particles into 1.5-ml microcentrifuge tubes, respectively.

(10).Centrifuge the two tubes at 16,110g at room temperature (23–25 °C) on a benchtop centrifuge for 15 min.


Autocatalytic reaction system:

(1).Harpin reaction: total volume 10ul <html> <body> <table width="200" border="1" cellspacing="2" cellpadding="1" background-color: #99CCFF> 

 <tr>
   <th scope="row"><span class="ys01">Water</span></th>
   <td><span class="ys01">7μL</span></td>
 </tr>
 <tr>
   <th scope="row"><span class="ys01">10×buffer</span></th>
   <td><span class="ys01">1μL</span></td>
 </tr>
 <tr>
   <th scope="row"><span class="ys01">10μMDNA</span></th>
   <td><span class="ys01"> 2μL</span></td>
 </tr>

</table> </body> </html>

Reaction condition:

(a)heating at 90°C for 5 minutes.

(b)Cooling on ice for 1 minute.

(c)At room temperature for 30 minutes before use.

The hairpins A B were prepared in reaction buffer as follows:

10×buffer:

Material Concentration(mM) MW(g/mol) Mass(g)
MgCl2·6H2O 4 203 0.8132
KCl 15 74.55 1.11825
Tris-HCl 10 121.14 1.2114

(2).reaction for A and I:

Mix 2μL A(at 2μM) and 2μL I(at 2μM), heating at 95°C for 5 minutes followed by cooling to room temperature over approximately 2.5 hours.

(3).reaction for B+(AI)： Mix 2μl B (at 2μM) and 2μl (AI) to react at room temperature for 15 minutes.

PAGE Gel:

Content Volume

Page Gel 30% 15ml

5 x TBE 6ml

ddH2O 9ml

TEMED 20µl

ASP 150µl

Total 30ml

prepare gel according the volumes in the table above

Pour gel into casting tray, and insert comb (make sure there are no bubbles)

Put the whole setup in 37℃ incubator for 30 min

Fill in TBE-Buffer

Prepare the sample by adding 6× loading buffer containing Golden View

Run the gel by 200 V and 30 min

Agarose Gel:

Measure 3g for 3% gel agarose in 250mL flask.

Add 100 mL 1X TBE into flask.

Microwave for about 3 minutes to disolve the powder.

Gently swirl in an ice water bath until it is not hot for your hand

Add EB stain in the gel with concentration of 5ul per 100ml

Pour gel into casting tray, and insert comb

cast the gel and cool the whole setup in room temperature

Prepare the sample by adding 6× loading buffer containing Golden View

Set to 150 V, run for 30 minutes


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