ds_94_4 {ruijter}

94-replicates-4-dilutions set

Description

The 94-replicates-4-dilutions set is a dilution series consisting of four 10-fold serial dilution points, starting from 15,000 molecules down to 15 molecules, created using 10 ng/µl yeast tRNA as carrier (Roche). The same dilution of the carrier was used to create the NTC sample.

qPCR was done on a CFX 384 instrument (Bio-Rad). A 384-well qPCR plate was prepared using a 96-well head pipetting robot (Tecan Freedom Evo 150). qPCR amplifications were performed in 8 µl containing:

• 4 µl iQ SYBR Green Supermix (Bio-Rad);

• 0.4 µl forward and 0.4 µl reverse primer (5 µM each);

• 0.2 µl nuclease-free water;

• 3 µl of standard oligonucleotide.

A total of 94 replicated reactions were dispensed for each of 4 dilution points. In addition, the NTC reaction was analyzed in 8 replicates.

All reactions were performed in 384-well plates (Hard-Shell 384-well microplates and Microseal B clear using adhesive seals (Bio-Rad).

The cycling conditions were comprised of 3 min polymerase activation at 95 °C and 45 cycles of 15 s at 95 °C and 30 s at 60 °C followed by a dissociation curve analysis from 60 to 95 °C.

Admonition

The concentration information for the curves in this data set was seemingly incorrectly indicated (confirmed by email with the senior author Prof. Jo Vandesompele). The annotation provided in the supplementary material that accompanies the original publication was reversed. So, to correct for this, the annotation of the number of copies in ds_94_4 has been changed:

• 15 became 15,000

• 150 became 1,500

• 1,500 became 150

• 15,000 became 15

Usage

ds_94_4

Format

ds_94_4

A data frame with 17,280 rows and 9 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".

target

Target identifier: MYCN.

sample_type

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

copies

Standard copy number: either 0 (for NTCs), or 15, 150, 1500 or 15000 for standards.

dilution

Dilution factor (the reciprocal of copies). Higher number means greater dilution.

cycle

PCR cycle.

fluor

Raw fluorescence values.

Source

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