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Team:TU Eindhoven/RCA/Attaching
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<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> | <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> | ||
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<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. | <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. | ||
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Revision as of 09:23, 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.
