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Team:KU Leuven/Project/Glucosemodel/qPCR
From 2013.igem.org
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| + | <h3>Why qPCR?</h3> | ||
<p align="justify">We perform this qPCR for two main reasons:</p> | <p align="justify">We perform this qPCR for two main reasons:</p> | ||
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| - | + | <h3>Step One</h3> | |
<p align="justify">We first started with the sample preparation, as described in the <a href="https://2013.igem.org/Team:KU_Leuven/Protocols#qRT-PCR_Protocol> qPCR protocol </a>. For this experiment we used our regular E. coli strains (DH5α), harbouring our <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060003">methyl salicylate brick</a> and used 3 biological repeats.<br/> | <p align="justify">We first started with the sample preparation, as described in the <a href="https://2013.igem.org/Team:KU_Leuven/Protocols#qRT-PCR_Protocol> qPCR protocol </a>. For this experiment we used our regular E. coli strains (DH5α), harbouring our <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060003">methyl salicylate brick</a> and used 3 biological repeats.<br/> | ||
After preparing three samples we started with the isolation of RNA. We then used the Nanodrop to get to know the concentration of our RNA and got the following results:</p> | After preparing three samples we started with the isolation of RNA. We then used the Nanodrop to get to know the concentration of our RNA and got the following results:</p> | ||
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| + | <h3>Step two</h3> | ||
<p align="justify">In a next step we removed DNA contamination by doing an extra DNase (Turbo DNase, Ambion) step as described in our protocol.<br/> | <p align="justify">In a next step we removed DNA contamination by doing an extra DNase (Turbo DNase, Ambion) step as described in our protocol.<br/> | ||
After the DNase step we needed to test whether there still is any DNA contamination in our RNA prep. We definitely have to remove all DNA contamination because the qPCR reaction will not differentiate between the original copy (gene on the plasmid) and the cDNA (Reverse Transcribed from the mRNA). If the original copy is still present in the sample, this will lead to overestimation of the quantity of mRNA molecules, and thus give false results. <br/> | After the DNase step we needed to test whether there still is any DNA contamination in our RNA prep. We definitely have to remove all DNA contamination because the qPCR reaction will not differentiate between the original copy (gene on the plasmid) and the cDNA (Reverse Transcribed from the mRNA). If the original copy is still present in the sample, this will lead to overestimation of the quantity of mRNA molecules, and thus give false results. <br/> | ||
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<img src="https://static.igem.org/mediawiki/2013/9/9e/Qrtpcr_3.png" alt="qrtpcr3"/> | <img src="https://static.igem.org/mediawiki/2013/9/9e/Qrtpcr_3.png" alt="qrtpcr3"/> | ||
<p align="justify">Fig 1. DNA gel electrophoresis after a PCR that amplifies a genomic region (csrA). </p> | <p align="justify">Fig 1. DNA gel electrophoresis after a PCR that amplifies a genomic region (csrA). </p> | ||
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Revision as of 19:22, 1 October 2013
Secret garden
Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!
- A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
- For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
- We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?
Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!
Why qPCR?
We perform this qPCR for two main reasons:
- With a qPCR we can check if our genes of interest are properly transcribed. This is a good characterisation of the methyl salicylate brick (BBa_K1060003)./li>
- We would like to know the amount of transcripts as an input for our methylsalicylate model.
Step One
We first started with the sample preparation, as described in the methyl salicylate brick and used 3 biological repeats.
After preparing three samples we started with the isolation of RNA. We then used the Nanodrop to get to know the concentration of our RNA and got the following results:
| ng/µl | 260/280 | 260/230 | |
|---|---|---|---|
| Sample A | 100,3 | 2,19 | 1,42 |
| Sample B | 56,3 | 2,17 | 0,78 |
| Sample C | 92 | 2,17 | 1,28 |
These results are satisfactory. Only the 260/230 values are lower than expected, but after purification these values will rise.
Step two
In a next step we removed DNA contamination by doing an extra DNase (Turbo DNase, Ambion) step as described in our protocol.
After the DNase step we needed to test whether there still is any DNA contamination in our RNA prep. We definitely have to remove all DNA contamination because the qPCR reaction will not differentiate between the original copy (gene on the plasmid) and the cDNA (Reverse Transcribed from the mRNA). If the original copy is still present in the sample, this will lead to overestimation of the quantity of mRNA molecules, and thus give false results.
To test whether there still is any DNA contamination we used PCR. We first tested if there is still any genomic DNA present in our sample by choosing a primer set on a conserved region. In our case we chose for the csrA gene. As a positive control we used genomic DNA from a MG1655 strain. After gelelectrophoresis we got the following results.
Fig 1. DNA gel electrophoresis after a PCR that amplifies a genomic region (csrA).
