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Team:TU Eindhoven/RCA/RCA on Cells
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/6/6d/TU_Eindhoven_RCA7.jpg" width="450" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;"> | <img id='Fig1' src="https://static.igem.org/mediawiki/2014/6/6d/TU_Eindhoven_RCA7.jpg" width="450" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;"> | ||
| - | <figcaption style="font-size:18px;color:#CCCCCC;">Figure 1. Bar graph showing median fluorescence after | + | <figcaption style="font-size:18px;color:#CCCCCC;">Figure 1. Bar graph showing median fluorescence after <br> different times of incubating for Rolling Circle Amplification. <br> The median is preferred over the average because of the logarithmic scale used in FACS measurements. <br> This graph clearly shows that longer incubation times increases the number of binding sites for a (fluorescent) complementary primer, thus demonstrating successful RCA on the cell membrane.</figcaption> |
</figure> | </figure> | ||
Revision as of 21:27, 16 October 2014
Rolling Circle Amplification on Cells
As mentioned in the introduction, with Rolling Circle Amplification a long piece of single strand DNA is generated by repeating the sequence of a circular strand multiple times. Since all necessary parts are now available to test this out on clickable cells that is what was done next. Cells expressing COMPx were reacted with the DBCO coupled DNA after which the circular template was added. Rolling Circle Amplification was then carried out with the Phi29 polymerase. Finally, to verify longer strands were indeed formed for longer reaction times a fluorescent probe was added to the samples. Relative fluorescence was then measured using FACS.
Summary
different times of incubating for Rolling Circle Amplification.
The median is preferred over the average because of the logarithmic scale used in FACS measurements.
This graph clearly shows that longer incubation times increases the number of binding sites for a (fluorescent) complementary primer, thus demonstrating successful RCA on the cell membrane.
To test if Rolling Circle Amplification works on cells, cells expressing COMPx were grown according to protocol . These were reacted for 1 hour at 4 °C and 500 rpm with the DBCO coupled primer. These were then spin purified and washed with PBS to remove any remaining primer. 1 sample was stored for use a control. Three remaining samples were incubated with circular, template, 5 mM DNTP-mix, Phi 29 polymerase and commercially supplied reaction buffer. Sample 1 was allowed to react for one hour before aphidicolin was added to stop the Phi29 polymerase, sample 2 for two and sample 3 for four hours. These samples were then again spin purified and washed with PBS.
All four samples and a blank without clicked DNA were incubated with a fluorescent complementary probe for 1 hour at 4 °C and 500 rpm. These were then spinned down to remove any unbound fluorescent probe. Samples were then resuspended in PBS and ran through the FACS. Figure 1 shows the median fluorescence of the samples. The rise in fluorescence for longer amplification times indicates that Rolling Circle Amplification has been successfully carried out on the cell membrane of our cells.
