"
Team:TU Eindhoven/RCA/Attaching
From 2014.igem.org
| (10 intermediate revisions not shown) | |||
| Line 121: | Line 121: | ||
<div id="tooplate_wrapper"> | <div id="tooplate_wrapper"> | ||
| - | <div id="contentwiki" style="height: | + | <div id="contentwiki" style="height:1285px;"> |
<div class="panel" id="home"> | <div class="panel" id="home"> | ||
| Line 128: | Line 128: | ||
<h2>Attaching DNA to DBCO-PEG<sub>4</sub></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, Click Coli | + | <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, Click Coli 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> |
<figure style="float:right;margin-right:0;"> | <figure style="float:right;margin-right:0;"> | ||
| Line 138: | Line 138: | ||
<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. | ||
<br><br> | <br><br> | ||
| - | To attach the molecules which normally don’t react together, DBCO-PEG<sub>4</sub>-NHS ester was ordered as well as a 5’-amine with a twelve carbon spacer functionalized primer. These two molecules | + | To attach the molecules which normally don’t react together, DBCO-PEG<sub>4</sub>-NHS ester was ordered as well as a 5’-amine with a twelve carbon spacer functionalized primer. These two molecules were reacted for two hours at room temperature to form the product.</p> |
<figure style="float:left;margin-left:0;"> | <figure style="float:left;margin-left:0;"> | ||
| Line 152: | Line 152: | ||
<br><br> | <br><br> | ||
The resulting molecule can be used to click DNA to the cell surface which was used to initiate a Rolling Circle Amplification reaction on the cell. | The resulting molecule can be used to click DNA to the cell surface which was used to initiate a Rolling Circle Amplification reaction on the cell. | ||
| - | |||
</p> | </p> | ||
| + | <br /> | ||
| + | <p>For a complete protocol for this step click <a target="_blank" href="https://static.igem.org/mediawiki/2014/2/2f/TU_Eindhoven_Labelling_amine-modified_DNA_with_DBCO-PEG4-NHS_ester.pdf">here</a> the protocol for the PAGE gel can be found <a target="_blank" href="https://static.igem.org/mediawiki/2014/0/00/TU_Eindhoven_Casting_and_running_PAGE_gel.pdf">here</a>.</p> | ||
</div> | </div> | ||
Latest revision as of 01:35, 18 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, Click Coli 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.
DNA with 12C spaced primary amine. Used to form our
clickable DNA oligo.
Overview
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 were reacted for two hours at room temperature to form the product.
DNA-binding stain. Heavier products travel through gel slower,
this shows that the reaction was successful and a clickable
DNA oligo has been formed.
After the reaction was completed the product was analysed on a 15% PAGE gel. On the gel it clearly showed that the product after two hours was heavier, thus indicating that DNA had reacted with DBCO-PEG4-NHS ester.
The final step was to purify the product and remove any unreacted DBCO-PEG4-NHS ester. This was done through ethanol precipitation of DNA. Next, product concentration in 100 µL MilliQ water was determined. The final concentration was used to calculate the yield of this reaction step, which was around 65% after washing.
The resulting molecule can be used to click DNA to the cell surface which was used to initiate a Rolling Circle Amplification reaction on the cell.
For a complete protocol for this step click here the protocol for the PAGE gel can be found here.
