Difference between revisions of "Taqman"

Latest revision as of 15:16, 10 May 2017

Taqman Real-Time qPCR

Materials

• cDNA: see First Strand cDNA Synthesis (AB Kit) for details

• Thermo Scientific ASsolute Blue qPCR Mix Plus ROX Vial (#AB-4136/B)

• 384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)

• Taqman Gene Expression Assay Primers stored at -20

Plate Preparation

1. Book 2h on qPCR machine by signing up on the sheet in room 6041

2. Prepare cDNA and dilute in water in a 96 well plate. Typically, a 20x dilution of cDNA leaves enough to be detected.

3. Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.

4. Sketch out plate in your notes. Typically, rows are different primers while columns are different cDNA.

5. Calculate how many samples x how many replicates per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells needed for each primer.

6. Prepare master mix. For a 10 uL reaction, you will need 5 uL of Absolute Blue mix, 0.5 uL of Taqman primer, and 2.5 uL of RNase Free Water. Mix components into 1.5 mL tube. Be sure to make 10-20% extra for volume loss.

7. Using the repeater multichannel pipetter, put on 2 or 3 tips (depending on plate arrangement) and set pipette to the amount of samples needed. Dispense 8 uL per well.

8. Using multichannel pipetter, add 2 uL of prepared cDNA into each designated well. Be sure to dispense to the bottom of the well.

9. Once plate is complete, put optically clear cover on it using plastic square to ensure the edges are sealed. Do not leave fingerprints on the seal.

10. You can prep the plate ~3 hours beforehand, keeping at 4C until the machine is ready.

11. Immediately before the run spin the plate briefly (2 minutes at 4000 RPM) in the swinging bucket centrifuge.

Run Protocol

• Set up run using Thermo Cloud/QuantStudio or Roche LightCycler