PCR Crash Course Part II

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<p>If PCR can’t be used to detect RNA, and coronaviruses are RNA viruses; there is no DNA and PCR does not replicate RNA, how can we detefct SARS-Cov-2? </p>

If PCR can’t be used to detect RNA, and coronaviruses are RNA viruses; there is no DNA and PCR does not replicate RNA, how can we detefct SARS-Cov-2?

  1. use qPCR to detect DNA

  2. we know of a method of creating RNA from DNA

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What is RT-qPCR?

  • is reverse transcriptase-qPCR

  • Reverse transcription is an enzyme-mediated process by which complementary DNA (cDNA) molecules are created from RNA templates.

  • After reverse transcription is completed, qPCR can continue as explained previously using cDNA as the template.

<ul><li><p>is <strong>reverse transcriptase-qPCR</strong></p></li><li><p>Reverse transcription is an enzyme-mediated process by which<strong> complementary DNA (cDNA)</strong> molecules are created from RNA templates.</p></li><li><p>After reverse transcription is completed, qPCR can continue as explained previously using<strong> cDNA as the template.</strong></p></li></ul><p></p><p></p>
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Performing RT-qPCR

  1. sample (template)

  2. Luna Enzyme (reverse transcriptase)

  3. Primer Set

  4. Probe Set

  5. Luna Buffer (DNA polymerase)

<ol><li><p>sample (template)</p></li><li><p>Luna Enzyme (reverse transcriptase)</p></li><li><p>Primer Set</p></li><li><p>Probe Set</p></li><li><p>Luna Buffer (DNA polymerase) </p></li></ol><p></p>
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Performing qPCR-Sample Concentration

Great sample concentration=?

Earlier detection of amplification

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LoD (LoD)

The lowest concentration of target DNA that can be detected during amplification

<p>The lowest concentration of target DNA that can be detected during amplification</p>
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In Lab Experiment

Synthetic Human DNA (P1)

Synthetic SARS-COV-2 RNA (N1)