ddPCR™ Supermix for Residual DNA Quantification
Catalog #Description

1864037
1864038
1864039
1864040

ddPCR Supermix for Residual DNA Quantification, 2 ml (2 x 1 ml vials), 200 x 20 μl reactions
ddPCR Supermix for Residual DNA Quantification, 5 ml (5 x 1 ml vials), 500 x 20 μl reactions
ddPCR Supermix for Residual DNA Quantification, 25 ml (5 x 5 ml vials), 2,500 x 20 μl reactions
ddPCR Supermix for Residual DNA Quantification, 50 ml (10 x 5 ml vials), 5,000 x 20 μl reactions

For research purposes only.

Description
ddPCR Supermix for Residual DNA Quantification is a2x concentrated, ready-to-use reaction cocktail containing
all components — except primers, probe(s), and template —required for probe-based Droplet Digital™ PCR (ddPCR). The
mixture delivers maximum target specificity and fluorescence
amplitude and minimum droplet variability to ensure precise
target quantification for the detection of residual host cell DNA.
The hot-start feature of the polymerase in the supermix
enables partitioning of samples into droplets while keeping
the enzyme inactive at ambient conditions. The supermix has
been optimized to support the amplification and detectionof DNA targets using hydrolysis probe–based assays and iscompatible with the use of uracil N-glycosylase (UNG) for PCR
decontamination. UNG may be purchased from a licensed
supplier.
Storage and Stability
ddPCR Supermix for Residual DNA Quantification is stable at–20°C through the expiration date printed on the labels. Once
thawed, it can be stored at 4°C for up to 2 weeks. Repeatedfreezing and thawing of the supermix is not recommended.
Quality Control
ddPCR Supermix for Residual DNA Quantification is freeof contaminating DNase and RNase. Stringent specifications
are maintained to ensure lot-to-lot consistency.
Recommendations for Optimal Results■ Follow general guidelines and recommendations forDroplet Digital PCR (refer to the Droplet Digital PCRApplications Guide, bulletin 6407)■■■

 uggested input quantities of DNA sample are 10 fg–100 ng
S
per 20 µl reaction; add femtogram to picogram amounts of
DNA for assays targeting highly repetitive sequences, suchas long or short interspersed nuclear elements (LINE or SINE),
and picogram to nanogram amounts of DNA for assays
targeting single-copy gene sequences
 n assay-specific standard curve is required to convert targetA
copy number to mass concentration for assays targeting an
unknown copy number of LINE, SINE, or Alu-like sequences no template control (NTC) should be included to rule outAcross-contamination■

 erform DNA extraction for complex samples with highPsalt (≥1.0 M salt) or low pH (buffer ≤3.0 pH), and perform
proteinase K treatment for samples containing a highconcentration of protein (≥0.1 mg/ml) prior to preparing
the ddPCR reaction mix to improve amplification. Forless complex samples, pretreatment is not necessary
if the sample is diluted at least 25-fold and the protein
concentration in the final ddPCR reaction is ≤0.1 mg/ml

Required Equipment
The QX200™ Droplet Digital PCR System (catalog #1864001),
QX200 AutoDG™ Droplet Digital PCR System (#1864100),
QX600™ Droplet Digital PCR System (#17007769),
QX600 AutoDG Droplet Digital PCR System (#17008371), or
QX ONE™ Droplet Digital PCR System (#12006536) is required.
Refer to the QX200 Droplet Reader and QX Manager SoftwareStandard Edition User Guide and QX200 Droplet Generator
Instruction Manual (10000107223 and 10031907, respectively),
the Automated Droplet Generator Instruction Manual(10043138), or the QX ONE Droplet Digital PCR System and
QX ONE Software User Guide (10000116512) for ordering
information about consumables, such as oils, cartridges,gaskets, plates, and seals.
Reaction Setup
1. Thaw all components to room temperature. Mix
thoroughly by vortexing the tubes to ensure homogeneity
because a concentration gradient may form during –20°C
storage. Centrifuge to collect contents at the bottom ofthe tubes.
2.

 repare samples at the desired concentration beforeP
setting up the reaction mix.

3.

 repare the reaction mix for the number of reactions
P
needed according to the guidelines in Table 1. Assemble
all required components except the sample, dispense
equal aliquots into each reaction tube, and add thesample to each reaction tube as the final step.

ddPCR Supermix for Residual DNA Quantification

Table 1. Preparation of the reaction mix.
Component

Volume per Reaction, µl

Final Concentration

Table 2. Thermal cycling conditions.*Temperature,Cycling Step°C

Time

Numberof Cycles

25

3 min

1

Enzyme activation95

10 min1

Denaturation95

30 sec40

Annealing/extension

60

1 min**

40

Enzyme deactivation

9810 min

1QX600 or QX200 ddPCR
System

430 min

1QX ONE ddPCR System

25

1 min

12x ddPCR Supermix for
10
Residual DNA Quantification

1xTarget primers/probe

Variable

900 nM/250 nM

Hold(QX ONE ddPCR System only)

SampleVariable

10 fg–100 ng/reaction

RNase-/DNase-free water

Variable—Total volume*20—* For the Automated Droplet Generator, prepare 22 µl per reaction.

4.

5.

 ix thoroughly by vortexing the tubes. Centrifuge briefly
Mto ensure that all components are at the bottom of thereaction tubes.
 ransfer the reaction mix from the reaction tubes to theTappropriate ddPCR Cartridge as follows:■■■ or the QX600 or QX200 Droplet Digital PCR System,Fload 20 μl of each reaction mix into a sample well ofa DG8 Cartridge. Follow subsequent instructions as
specified in the QX200 Droplet Generator Instruction
Manual (10031907)

Hold* For the PTC Tempo Deepwell Thermal Cycler or C1000 Touch Thermal Cycler
with 96–Deep Well Reaction Module, use a heated lid set to 105°C and set thesample volume to 40 μl.
** Check/adjust ramp rate settings to ~2°C/sec.

Data Acquisition and Analysis
1. Follow instructions based on the system in use:■ or the QX600 or QX200 AutoDG Droplet Digital PCR
FSystem, follow instructions in the Automated Droplet
Generator Instruction Manual (10043138)■

 or the QX ONE Droplet Digital PCR System, load
F
20 μl of each reaction mix into a sample well of a
GCR96 Cartridge. Follow subsequent instructions as
specified in the QX ONE Droplet Digital PCR Systemand QX ONE Software User Guide (10000116512)■Thermal Cycling Conditions
Follow instructions based on the system in use:■■■ or the QX600 or QX200 Droplet Digital PCR System,
Fafter droplet generation with the QX200 Droplet Generator,
carefully transfer droplets into a clean 96-well plate. Seal theplate using the PX1 PCR Plate Sealer (#1814000) at 180°C for5 sec. Proceed to thermal cycling (see Table 2)
 or the QX600 or QX200 AutoDG Droplet Digital PCR
FSystem, remove the droplet plate containing ddPCR
droplets from the Automated Droplet Generator. Seal theplate using the PX1 PCR Plate Sealer at 180°C for 5 sec.
Proceed to thermal cycling (see Table 2)
 or the QX ONE Droplet Digital PCR System, thermalFcycling is integrated into and sequentially performed by thesystem itself. Hence, no additional equipment or sample
handling is required for this step. Refer to the QX ONE
Droplet Digital PCR System and QX ONE Software User
Guide (10000116512) for plate setup instructions.
Use appropriate thermal cycling conditions as specified
in Table 2

Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Drive, Hercules, CA 94547
510-741-1000

 or the QX600 Droplet Digital PCR System and theF
QX600 AutoDG Droplet Digital PCR System, refer tothe QX600 Droplet Reader and QX Manager SoftwareStandard Edition User Guide (10000153877)
 or the QX200 Droplet Digital PCR System and theF
QX200 AutoDG Droplet Digital PCR System, refer tothe QX200 Droplet Reader and QX Manager SoftwareStandard Edition User Guide (10000107223)
 the QX ONE Droplet Digital PCR System, referFor
to the QX ONE Droplet Digital PCR System and
QX ONE Software User Guide (10000116512) and the
QX ONE Software User Guide for Standard Edition(10000116655) or Regulatory Edition (10000116656)

2.

 fter data acquisition, select samples in the well selectorA
under Analyze. Examine the automatic thresholding
applied to the 1-D amplitude data and, if necessary, set
thresholds manually by applying either the single well or
multiwell threshold between the positive and negativedroplets. Use the NTC well to determine the appropriate
threshold for sample wells.

3.

The concentration reported is copies/µl of the final
1x ddPCR reaction.

Visit bio-rad.com/ddPCRRDQ for more information.
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10000162847 Ver B (10048259)

23-03600423