(r2) Barrick Lab :: PrimerEfficiencyqPCR

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*%RED% THIS PAGE IS CURRENTLY UNDER CONSTRUCTION%ENDCOLOR%*

_Goals_

   * Test that primers work
   * Determine the efficiency with which your primers bind to your target (This will be important when you do your final analysis)

_Before you begin_
   * The template for this qPCR will be the PCR product of each of your target genes. So before you get started you will need to perform PCR off of either genomic DNA or cDNA (this depends on how you designed your primers)
   * Run your PCRs like you normally would (use a 60C annealing temperature).
   * PCR purify your product and then dilute to a concentration of ~ 0.01ng/ul (trust me...it seems low but it works!)
   * Now you're good to go ahead and set up your qPCR reactions!

Typical plate setup for 3 different primers:

     <img src="%ATTACHURLPATH%/PrimerEffplate.png" alt="PrimerEffplate.png" width='757.33333' height='208.6666' />

(Numbers are PCR product dilution with "10" representing a 1:10 dilution of your 0.01ng/ul PCR product prepped as described above)

_Conditions_


_What you are looking for_




<<[[qPCR][Return to qPCR page]]

-- Main.KateElston - 21 Mar 2019

Barrick Lab > ProtocolList > QPCR > PrimerEfficiencyqPCR

Contributors to this topic

KateElston, DanielDeatherage

Topic revision: r2 - 2019-04-03 - 22:33:44 - Main.KateElston