QPCR

Real Time qPCR

Materials

• cDNA: see First Strand cDNA Synthesis (AB Kit) for details
• SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; Vendor Link)
• 384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)
• Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks- the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example- this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water). This primer mix can be stored at -20 in a Working Primers box. Design primers according to Primer Design for qPCR

Plate Preparation

1. Book 2h on qPCR machine by signing up on the sheet in room 7013 and on the google calendar
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 the plate in your notes. Typically rows are different primers while columns are different cDNA's
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 need for each primer.
6. Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 20-25% more than you need.
7. Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.
8. Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
9. Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.
10. Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal. Apply cover by starting in the middle of plate, working out towards edges, and finish by applying high pressure to edges, ensuring a proper seal.
11. You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.
12. Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

Run Protocol

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

Briefly, log into thermo cloud

• Select "Design and Analysis New, qPCR" from the home screen
• Select "set up plate"
• Select "comparative CT-SYBR"
• Correct volume to read, "10.0uL" instead of "20.0uL"
• Select "Plate set up"
• Navigate to the drop down menu marked "passive reference" and select "none."
• Then import plate setup with linked CSV (MUST be a csv file).
• Save to the cloud and upload on the machine when you insert the plate.

Calculations

see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations