1. qPCR experiment design

2. Experiment Absolute qPCR Relative qPCR Standard 1 Standard 2
NTC
Sample B
Standard 5
Standard 6
Ref1 St 1
Ref1 St 2
Ref1 St 3
Ref1 St 4
Ref2 St 1
Ref2 St 2
Ref2 St 3
Ref2 St 4
Ref3 St 1
Ref3 St 2
Ref3 St 3
Ref3 St 4
GOI St 1
GOI St 2
GOI St 3
GOI St 4
Ref1 Sample 1
Ref2 Sample 1
Ref3 Sample 1
GOI Sample 1
Ref1 Sample 2
Ref2 Sample 2
Ref3 Sample 2
GOI Sample 2

3. Target
Target = target gene = a gene you will detect with a certain primer pair
primers are designed to anneal on their target gene
If primers also anneal on another gene, it will cause off-target
(non-specific) amplification
Please write down what are the targets in this experiment: 1. 2. 3. 4. 5.

4. where else on the plate will it be?
Please use colours to mark reactions (plate wells) with the same target
Absolute
Relative
for example: Ref gene 1
where else on the plate will it be?
plate layout 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H
Reference gene 1

5. n of reactions for Absolute qPCR n of reactions for Relative qPCR
Please count the number of reactions (plate wells) with the same target
Target gene name
n of reactions for Absolute qPCR
n of reactions for Relative qPCR

6. Final concentration of each primer in the reaction should be 125 nM
Please write down premix composition for each target
Absolute
Relative
premix x1 x
2x master mix
10 ul
100 uM primer Fw
0,025 ul
100 uM primer Re
template
5 ul MQ
4,95 ul
You are welcome to dilute your primers stocks 20 or mote times to make pipetting more convenient.
If you do so, please recalculate primer and MQ volumes you will add to the reactions.
Final concentration of each primer in the reaction should be 125 nM

7. Please write down what are the templates used in this experiment:
Template = DNA you add to the PCR (e.g. genomic DNA, cDNA, plasmid etc.)
Please write down what are the templates used in this experiment: 1. 2. 3. 4. 5. …

8. Please mark reactions with exactly the same template
(use symbols or text. sic! in this case different dilutions do not count as exactly the same template)
Absolute
Relative
plate layout 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H

9. n of reactions for Absolute qPCR n of reactions for Relative qPCR
Please count the number of reactions with the exactly the same template
template name
n of reactions for Absolute qPCR
n of reactions for Relative qPCR

10. n of reactions for Absolute qPCR n of reactions for Relative qPCR
Please calculate the total volume of each template you will need
template name
n of reactions for Absolute qPCR
Volume in uL
n of reactions for Relative qPCR

11. What is the dilution factor you are going to use?
Absolute qPCR
What dilutions you will make for in this experiment and is the purpose of these dilutions?
What is the dilution factor you are going to use?
Please note, that for the sake of time you will not dilute the sample with unknown concentration for this experiment. Please remind yourself, why would it be beneficial to dilute it if running this type of experiment for the very first time?

12. Please write ul of each component for each dilution
and mention which components are used for what dilution
Absolute qPCR
sample to be diluted
ul of sample
ul of MQ
Standard curve dilution 1
Standard curve dilution 2
Standard curve dilution 3
Standard curve dilution 4
Standard curve dilution 5
Standard curve dilution 6

13. Relative qPCR What dilutions you will make for this experiment?
What is the purpose of these dilutions?
What is the dilution factor you are going to use for efficiency curves?
What will be the composition of your DNA pool for primer efficiency and why?
How much are you going to dilute your samples and why?

14. Please write ul of each component for each dilution
and mention which components are used for what dilution
Relative qPCR
sample(s)
ul of sample(s)
ul of MQ
DNA pool
DNA pool dilution 1
DNA pool dilution 2
DNA pool dilution 3
DNA pool dilution 4
sample 1
sample 2

15. Protocol (or PCR program)
what happens here?
1. 95 oC minutes
what happens here?
why not more or less?
2. 95 oC seconds
3. 60 oC seconds
40 repeats
(acquisition)
what happens here?
what is this for? why is it at this step?
4. 95 oC minute
what is this for? oC
acquisition after each 0.5 oC change
what happens here?
what is this for?