Team:KU Leuven/Project/Glucosemodel/qPCR

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We perform this qPCR for two main reasons:

  1. 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>
  2. We would like to know the amount of transcripts as an input for our methylsalicylate model.

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/µl260/280260/230
Sample A100,32,191,42
Sample B56,32,170,78
Sample C922,171,28

These results are satisfactory. Only the 260/230 values are lower than expected, but after purification these values will rise.
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.