qPCR

Objective: Take cDNA and perform a qPCR to quantify gene expression for 3 sets of samples amplified using 2 different primers

Primers

qPCR Protocol

Standard Operating Protocol (SOP)

Written 20150702 by Sam White.

Reagents:

• SsoFast EvaGreen Supermix (BioRad: 172-5203)
• Primer working stocks (10uM)
• DNase-free H2O (NanoPure H2O)

Personal Protective Equipment (PPE):

• Gloves

Equipment:

• Pipettes (10 - 1000uL)
• Filtered pipette tips
• White PCR plates, non-skirted, low profile (USA Scientific: 1402-9590)
• Optically clear strip caps (USA Scientific: 1400-3800)
• Sterile 1.7mL snap-cap microfuge tubes (Genesee: 22-281S)
• Real-time PCR machine
• ice

Procedure

Total Time: ~ 2.0 - 4.0hrs

Cost/sample: ~ $0.42

1. Read the manufacturer's protocol.
2. Read this protocol.
3. Verify sufficient quantities of reagents and samples before beginning.
4. Wear clean gloves.
5. Prepare master mix. Be sure to make a master mix volume that will accommodate the following: all of your samples, two water (i.e. no template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. A single reaction is shown below:

   Component	Volume(uL)	Final Concentration
   2x SsoFast EvaGreen Supermix	10	1x
   Forward Primer (10uM)	0.5uL	0.2uM
   Reverse Primer (10uM)	0.5uL	0.2uM
   Water	Variable	Use to bring reaction volume up to 20uL (including template)

6. Distribute appropriate amount of master mix (volume of master mix + template = 20uL) to white PCR plate.
7. Add template.
8. Cap with optical PCR caps.
9. Spin plate for 1min @ 3000g.
10. Put plate in real-time PCR machine.
11. Recommended cycling parameters (40 cycles) are listed below. They may need to be changed to accommodate your specific primers/samples. See manufacturer's protocol for recommendations.
    Step 1 - 98C 2mins
    Step 2 - 98C 5secs
    Step 3 - 60C 5secs
    Step 4 - Plate read
    Step 5 - Got to Step 2 39 more times
    Step 6 - Melt curve 65C - 95C, increment 0.5C, wait 2secs, plate read.

Calculations:

PCR PLATE

Sample Definitions: 

1-1, 1-2, 2-1, 2-2 Samples taken originally for sequencing work added them to compare to samples taken at a later date to check for variation due to time in captivity,

Day 1-4 are samples taken during manipulation experiment where animals were under artifical lighting to simulate day time hours.

Night 1-4 animals left in the dark for two hours to simulate a night time environment.

Results

qPCR calculations

Primer set 1

Quantitation

Link to data:
https://docs.google.com/spreadsheets/d/1XJRXXuiJhDiIlJ-Br6-g-lJjVEifGOmaOubv4rrRj6M/edit?usp=sharing

Melting Curve

Primer Set 2

Quantitiation

Melting Curve

Note: realized that I had forgotten my no template controls when doing the experiment will rerun qPCR to confirm that data is valid.