Covid-19 is actually an RNA virus. Discuss what extra step(s) you need to do in order for your PCR based detection of the virus to be successful

 

Go to the primer design website and register for an account:

Primer design website:

https://www.idtdna.com/PrimerQuest/

(free after registration)

 

Exercise:  download the Covid-19 N protein nucleotide sequence here:

https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2?from=28274&to=29533&report=fasta

 

 

copy the sequence into the idtdna.com website and design your own primers against Covid-19.  Select regular PCR (PCR – 2 primers).  You may leave all other parameters at the default settings.

Select any set of primers from the website.  Click on any of the primer sets and click “assay details”  and do a printout of the screen showing the sequence of the forward primer, reverse primer,  and the sequence of the Covid-19 N protein and save that as a PDF file.

 

Upload the PDF file together with your assignment.   You may also just copy and paste that page directly into this word file.

 

After you have chosen the primers, copy and paste the sequence of the primer into the NEB website to determine what’s the best enzyme Annealing temperature determination for your PCR reaction:

 

 

https://tmcalculator.neb.com/#!/main

 

You may choose any of the commonly used PCR enzymes (I personally prefer Taq or Vent but others would work too).  Note the annealing temperature would be different depending on what enzymes you are using.

 

Save the NEB screen showing the the Tm of the primers, the enzyme you have chosen and the suggested annealing temperature

 

(20 points for the pictures)

 

 

 

 

Answer ALL of the following questions:

 

  1. Write out all the steps you need to do your own PCR test as follows:
  2. All Reagents needed: (5 points)

 

 

 

 

 

 

 

  1. Temperature programming of the thermo cycler for the primers and enzyme you have chosen. (10 points)

 

 

 

 

 

 

  1. Comments on what may happen if you set the annealing temperature (a) too low and (b) too high. (5 points)

 

 

 

 

 

  1. Covid-19 is actually an RNA virus. Discuss what extra step(s) you need to do in order for your PCR based detection of the virus to be successful    (5 points)

 

 

 

 

 

 

  1. In a hypothetical PCR process, primer 1 has Tm of 55ºC and primer 2 has Tm of 60º Discuss what annealing temperature you would set your PCR machine for the PCR reaction to be successful.  Provide a brief explanation of your choice   (5 points)

 

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