q PCR experiment design Experiment Absolute q PCR
q. PCR experiment design
Experiment Absolute q. PCR Relative q. PCR Ref 1 Sample 1 Standard 1 Ref 1 St 1 Ref 3 St 1 Standard 2 Ref 1 St 2 Ref 3 St 2 Ref 2 Sample 1 Standard 3 Ref 1 St 3 Ref 3 Sample 1 Standard 4 Ref 1 St 4 Ref 3 St 4 GOI Sample 1 Standard 5 Ref 2 St 1 GOI St 1 Ref 1 Sample 2 Standard 6 Ref 2 St 2 GOI St 2 Ref 2 Sample B Ref 2 St 3 GOI St 3 Ref 3 Sample 2 NTC Ref 2 St 4 GOI Sample 2
Target = target gene = a gene you will detect with a certain primer pai 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.
Please use colours to mark reactions (plate wells) with the same target Relative Absolute for example: Ref gene 1 where else on the plate will it be? plate layout 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 Reference gene 1
Please count the number of reactions (plate wells) with the same target Target gene name n of reactions for Absolute q. PCR n of reactions for Relative q. PCR
Please write down premix composition for each target Absolut e premix x 1 2 x master mix x Relative x x 10 ul 100 u. M primer 0, 025 ul Fw 100 u. M primer 0, 025 ul 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 n. M
Template = DNA you add to the PCR (e. g. genomic DNA, c. DNA, plasmid etc. ) Please write down what are the templates used in this experiment: 1. 2. 3. 4. 5. …
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) Relative Absolute plate layout 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12
ease count the number of reactions with the exactly the same template name n of reactions for Absolute q. PCR n of reactions for Relative q. PCR
Please calculate the total volume of each template you will need template name n of reactions for Absolute q. PCR Volume in u. L n of reactions for Relative q. PCR Volume in u. L
Absolute q. PCR hat dilutions you will make for in this experiment and is the purpose of these dilution 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?
Please write ul of each component for each dilution and mention which components are used for what dilution Absolute q. PCR Standard curve dilution 1 Standard curve dilution 2 Standard curve dilution 3 Standard curve dilution 4 Standard curve dilution 5 Standard curve dilution 6 sample to be diluted ul of sample ul of MQ
Relative q. PCR 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?
Please write ul of each component for each dilution and mention which components are used for what dilution Relative q. PCR DNA pool dilution 1 DNA pool dilution 2 DNA pool dilution 3 DNA pool dilution 4 sample 1 sample 2 sample(s) ul of MQ
Protocol (or PCR program) what happens here? 1. 95 o. C 7 minutes what happens here? 2. 95 o. C 3. 60 o. C why not more or less? 10 seconds 40 repeats (acquisition) 30 seconds what happens here? what is this for? why is it at this step? 4. 95 o. C 1 minute what is this for? 5. 60 -95 o. C acquisition after each 0. 5 o. C change what happens here? what is this for?
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