ds_competimer {ruijter}

Competimer set

Description

Competitive primers were synthesized on basis of identical sequence and blocked by amination at the 3' end to allow annealing, but avoid elongation during the PCR process.

A six-point 4-fold serial dilution series made from reference human genomic DNA (Roche), starting from 64 ng/µl down to 0.0625 ng/µl, was created in 10 ng/µl yeast tRNA as carrier. The same dilution of the carrier was used to create a NTC sample.

qPCR amplifications were performed in 7.5 µl total reaction volume containing:

• 3.75 µl 2x custom made qPCR SYBR green I Mastermix (Eurogentec)

• 0.375 µl forward primer (5 µM) and 0.375 µl reverse primer (5 µM)

• 1 µl of a mixture of nuclease-free water and equal amounts of both forward and reverse competitive (aminated) primers

• 2 µl diluted standard

A total of 7 competitive mixes were prepared for each dilution point, containing 0%, 5%, 10%, 20%, 30%, 40%, and 50% (of the total amount of primer) competitive (aminated) forward and reverse primers. Each reaction was run in triplicate.

The qPCR cycling was performed on the LightCycler480 (Roche) using white LightCycler480 384-multiwell plates with Light-Cycler480 sealing foils (Roche).

The cycling conditions were com prised of 10 min polymerase activation at 95 °C, and 45 cycles of 15 s at 95 °C, 30 s at 60 °C, followed by a dissociation curve analysis from 60 to 95 °C.

Usage

ds_competimer

Format

ds_competimer

A data frame with 6,615 rows and 10 columns:

well

Well identifier.

replicate

Replicate identifier.

dye

In all reactions the SYBR Green I master mix (Roche) was used, so the value is always "SYBR".

pct

Percentage of competitive (aminated) primers in the mix.

conc

Concentration of reference human genomic DNA (Roche): from 64 ng/µl down to 0.0625 ng/µl.

target

Target identifier: AluSx.

sample_type

Sample type: "ntc" (no template control) or "std" (standard).

dilution

Dilution factor (the reciprocal of conc, i.e. 64 / conc). Higher number means greater dilution.

cycle

PCR cycle.

fluor

Raw fluorescence values.

Source

https://medischebiologie.nl/wp-content/uploads/2019/02/qpcrdatamethods.zip