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Okey dokey, let’s get this thread on the road. We’re going to sequence the viral genomes of a bunch of COVID-19 positive controls (ie not infectious, just inactivated viral RNA). This is aimed at the general public rather than scientists. 1/n
The first thing we have to do is to create DNA from the viral RNA. This is so the next step (PCR) will work. This is called reverse transcription and uses an enzyme obviously called reverse transcriptase. 2/n
In order to create the DNA from the viral RNA (it’s called cDNA) we have to add the reverse transcriptase and a buffer to the same and then incubate it for 10 minutes at 55C and then 1min at 95C to kill the enzyme. It’s just like cooking, but smaller. 3/n
While we wait for the cDNA incubation to finish, meet Bob. Bob is one of the @taxagenomics pipetting robots and until recently was the powerhouse of COVID-19 testing on the Isle of Man, isolating RNA from patient samples. 4/n
Now that we’ve created some cDNA we need to set up the PCR reactions. PCR is how we amplify the tiny amount of cDNA we have and make enough of it to sequence. It’s like a molecular photocopier. We also need to mix small volumes so here’s the whirly mixer (technical term) 5/n
We mix the right volumes and concentrations of these reagents to put all the nuts and bolts of the PCR reaction together. It doesn’t look like much once the samples are added. Now for more cooking. 6/n
The PCR reactions for COVID sequencing are “multiplex” which means there are many individual PCR reactions happening in the same tube. For this protocol it takes about 5 hours, so we leave it to work overnight because who has time to watch a boiling kettle? More tomorrow! 7/n
