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Team:TU Eindhoven/RCA/Attaching
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| - | <h2> | + | <h2>Attaching DNA to DBCO-PEG<sub>4</sub></h2> |
| - | + | ||
| - | < | + | <p>For the Rolling Circle Amplification to be used on the surface of cells a DNA-primer has to be covalently linked to the cells. This primer can then be used to start the amplification of the DNA-strand to form the long strands needed for Multiple Chain Amplification. In order to attach DNA to the cell surface, the DBCO-Azido click system developed was used. The steps described focus on attaching a short DNA oligo to a DBCO-PEG<sub>4</sub> linker molecule. This is the building block that is used to click DNA to the cells and start a Rolling Circle Amplification reaction.</p> |
| - | < | + | |
| - | < | + | <h3>Summary</h3> |
| - | <img id=' | + | <figure style="float:right;margin-right:0;"> |
| - | < | + | <img id='Fig1' src="https://static.igem.org/mediawiki/2014/f/f0/TU_Eindhoven_RCA5.jpg" width="500" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;"> |
| + | <figcaption style="font-size:18px;color:#CCCCCC;"></figcaption> | ||
| + | </figure> | ||
| - | <p> | + | <p>In order to attach DNA-primers to a cell the click-system developed by Team Eindhoven 2014 can be used. For this to work the primer needs to be functionalized with a DBCO-group. This was done by attaching a DBCO-PEG<sub>4</sub> molecule to the oligo required for Rolling Circle Amplification. |
| - | + | <br><br> | |
| - | < | + | To attach the molecules which normally don’t react together, DBCO-PEG4-NHS ester was ordered as well as a 5’-amine with a twelve carbon spacer functionalized primer. These two molecules where reacted for two hours at room temperature to form the product. |
| - | < | + | </p> |
<h4>Bibliography</h4> | <h4>Bibliography</h4> | ||
Revision as of 09:22, 15 October 2014
Attaching DNA to DBCO-PEG4
For the Rolling Circle Amplification to be used on the surface of cells a DNA-primer has to be covalently linked to the cells. This primer can then be used to start the amplification of the DNA-strand to form the long strands needed for Multiple Chain Amplification. In order to attach DNA to the cell surface, the DBCO-Azido click system developed was used. The steps described focus on attaching a short DNA oligo to a DBCO-PEG4 linker molecule. This is the building block that is used to click DNA to the cells and start a Rolling Circle Amplification reaction.
Summary
In order to attach DNA-primers to a cell the click-system developed by Team Eindhoven 2014 can be used. For this to work the primer needs to be functionalized with a DBCO-group. This was done by attaching a DBCO-PEG4 molecule to the oligo required for Rolling Circle Amplification.
To attach the molecules which normally don’t react together, DBCO-PEG4-NHS ester was ordered as well as a 5’-amine with a twelve carbon spacer functionalized primer. These two molecules where reacted for two hours at room temperature to form the product.
Bibliography
Mazutis, Linas, John Gilbert, W Lloyd Ung, David A Weitz, Andrew D Griffiths and John A Heyman. Single-cell analysis and sorting using droplet-based microfluidics. Nature protocols 8.5 (2013): 870-891.
