User guide

Quantitation methods
Before performing digital PCR experiments, consider quantifying the amount of
gDNA or cDNA in each sample.
We recommend the following methods of quantitation:
• Quant-iT
™
assay nucleic acid quantitation using the Qubit
®
Quantitation Platform
or
• Use spectrophotometer to determine nucleic acid concentration
Should a target be present at a sufficiently high concentration in the sample of
interest, it is possible that all reaction replicates will be positive, thus preventing the
determination of the target concentration. In this case, the sample must first be diluted
prior to running the digital PCR experiment.
In a digital PCR experiment performed on a QuantStudio
®
3D Digital PCR System,
gDNA samples are diluted down to a limiting quantity, such that most individual
PCR reactions contain either zero or one target molecule. The procedure for
determining the optimal dilution for a sample differs depending on whether or not
the target copy number per genome is known.
If the target copy number per genome of your samples is known, dilute the samples
so that, when loaded on a QuantStudio
®
3D Digital PCR 20K Chip, each through-hole
reaction will contain approximately 0.6 to 1.6 copies of the target sequence. For
example, assuming 3.3 pg/copy of a given gene are present per human genome and a
865-pL reaction well volume, the stock gDNA in a given sample would be diluted
down to 600 copies/μL or 1.98 ng/μL in the final reaction to give 0.6 copies per
reaction well.
How to determine the target copy number per genome
To help determine copy number per genome, collect the following information:
1.
If the source or species of the gDNA is known but the genome size of the
organism of interest is unknown, refer to http://www.cbs.dtu.dk/databases/
DOGS/index.html to determine the size of the genome in question.
2.
Once the size of the genome is known, determine the mass of the genome using
the following formula:
m = ( n ) ( 1.096 × 10
-21
g/bp )
where m is the genome mass in grams, and n is the genome size in base pairs.
The following example calculates the mass of the human genome using the Celera
Genomics estimate of 3.0 × 10
9
bp (haploid):
m = (3.0 × 10
9
bp) (1.096 × 10
-21
g/bp)
m = 3.3 × 10
-12
g or 3.3 pg
The example is relevant to any gene that is present at the “normal” rate of two copies
per diploid genome, such as RNase P, and provides a basis to perform a digital
screening experiment to determine the optimal digital range.
Sample dilution
Determine the
optimal dilution
when the target is
known
Chapter 2 Prepare Samples and Load Reactions
Prepare the DNA samples
2
QuantStudio
®
3D Digital PCR System User Guide 37