4.7.4. Two-step Quantitative PCR for high-throughput assays

The below variant of qPCR is for a 96-well plate format on the CFX96 real time system (BioRad) or related machines, and works for both bee transcripts and pathogen targets. The primary difference over the prior protocol is that this one is initiated with cDNA generated in a non-specific way, rather than from de novo reverse-transcription for each viral and/or host test and control (as shown in the previous section).

  1. Mix 1x SsoFast EvaGreen® supermix (BioRad) with 3 mM of each forward and reverse primer for a given target (final volume 4 µl).
  2. Add 1 ml (~8 ng) of cDNA template to specific wells.
  3. Use the following cycling conditions:
    97°C for 1 min,
    45 (maximum 50) cycles of:
    95°C for 2 sec,
     60°C for 5 sec,
    Melt curve from 65-95°C at +0.5°C/5 sec increments.
  4. Verify amplicon melting points for every positive sample. 
    Amplicons from positive controls and initial samples should be cloned into pGEM-T Easy vector (Promega) to verify sequence.
  5. Run four distinct no-template controls on the plate to monitor for contamination and non-specific amplification.
  6. Standard curves should be run using a recombinant plasmid dilution series of the primer targets from 101 to 108 copies, providing a linear equation to calculate the copy number in each sample using 10 Cq – b / m, where Cq = quantification cycle, b = y-intercept, and m = slope.

 

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