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Experimenttal part. Ref2 Sample 1. Ref1 Sample 1. Ref3 Sample 1. Ref3 Sample 2. Ref1 Sample 2. Ref2 Sample 2. GOI Sample 1. GOI Sample 2. Standard 6. Standard 5. Standard 4. Standard 2. Standard 1. Standard 3. Ref1 St 4. Ref2 St 3. Ref1 St 1. Ref1 St 2. Ref1 St 3.
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Ref2 Sample 1 • Ref1 Sample 1 • Ref3 Sample 1 • Ref3 Sample 2 • Ref1 Sample 2 • Ref2 Sample 2 • GOI Sample 1 • GOI Sample 2 • Standard 6 • Standard 5 • Standard 4 • Standard 2 • Standard 1 • Standard 3 • Ref1 St 4 • Ref2 St 3 • Ref1 St 1 • Ref1 St 2 • Ref1 St 3 • Ref2 St 1 • Ref3 St 1 • Ref3 St 2 • Ref3 St 3 • Ref3 St 4 • Ref2 St 4 • Ref2 St 2 • GOI St 3 • GOI St 4 • GOI St 2 • Sample B • NTC • GOI St 1 • Experiment • Relative qPCR • Absolute qPCR • you need to provide: • 1. RNA or cDNA from two samples you want to compare (sample 1 and sample 2) • 2. sequences of primers for one gene of interest (GOI) and three reference genes • you will need to: • estimate efficiency of your primers • find out the amount of your GOI in sample 1 compared to sample 2 • you will be provided with: • 1. sample A: plasmid of known concentration • 2. forward and reverse primers to detect the plasmid • 3. %E of the primers • 4. sample B:the same plasmid with unknown concentration • you will need to: • find out the concentration of the sample B
Kits and Protocols For qPCR we will use DyNAmo Flash SYBR Green qPCR Kit (# F-415L, ThermoScientific.follow the link for Manual and other info) • For RT we will use Maxima First Strand cDNA Synthesis Kits for RT-qPCR (# K1642, ThermoScientific. follow the link for Manual and other info)
Absolute qPCR • Use sample A to make 6 serial 10 fold dilutions. • Take into consideration: • each dilution will be checked in 3 identical PCR reactions (technical replicates, a control for pipetting error) • you will use 5 ul of template for each PCR reaction • sample A: 50 ul of plasmid, concentration 10 ng/ul • 5 uM forward primer (F) • 5 uM reverse primer (R) • sample B: the same plasmid, concentration is unknown • NTC= no template control= water • 2. Calculate the amount of qPCR reactions required and prepare corresponding volume of the Master mix (amount of reactions + 2) • 3. Pipet the Master mix into the plate (15 ul /well) • 4. Add assigned templates to the Master mix in the wells • 5. Seal the plate with the film. store at 4oC before run • 6. Program the run • 7. Perform the run • 8. Your data file will be uploaded on the course web site • 9. Analyse the data • 10. Write a report • 11. Make a presentation
Relative qPCR • 1. Pool together x+y ul of our cDNA samples. If necessary dilute it up to 30 times (call this sample ”cDNA pool”). • Prepare a four serial dilutions of the cDNA pool • Take into consideration: • each dilution will be checked in 3 identical PCR reactions (technical replicates, a control for pipetting error) • you will use 5 ul of template for each PCR reaction • try not to dilute your initial cDNA samples more than 300 times • sample 1: cDNA of your choice • sample 2: another cDNA of your choice • 5 uM Ref1 primers (forward and reverse) • 5 uM Ref2 primers (forward and reverse) • 5 uM Ref3 primers (forward and reverse) • 5 uM GOI primers (forward and reverse) • 2. Calculate the amount of qPCR reactions required and prepare corresponding volume of the Master mix (amount of reactions + 2) • 3. Pipet the Master mix into the plate (15 ul /well) • 4. Add assigned templates to the Master mix in the wells • 5. Seal the plate with the film. store at 4oC before run • 6. Program the run • 7. Perform the run • 8. Your data file will be uploaded on the course web site • 9. Analyse the data • 10. Write a report • 11. Make a presentation
