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 with minimum droplet variability to ensure precise
target quantification for the detection of residual host cell DNA.
The hot-start features of the polymerase enable partitioningof samples into droplets while keeping the enzyme inactiveat ambient conditions. The supermix has been optimized tosupport the amplification and detection of DNA targets usinghydrolysis probe–based assays and is compatible with the useof 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 PCR
Applications 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 necessaryif 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), or
QX ONE Droplet Digital PCR System (#12006536) is required.
Refer to the QX200 Droplet Reader and QuantaSoft Softwareand QX200 Droplet Generator Instruction Manuals (10031906
and 10031907, respectively), the Automated Droplet Generator
Instruction Manual (10043138), or the QX ONE Droplet Digital
PCR System Instruction Manual (10000116512) for orderinginformation 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 ofeach tube.
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.

Table 1. Preparation of the reaction mix.
Component

Volume per Reaction, µl

2x ddPCR Supermix for
10Residual DNA Quantification

Final Concentration
1x

Target primers/probe

Variable

900 nM/250 nM

SampleVariable

10 fg–100 ng/reaction

RNase-/DNase-free water

Variable—

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

ddPCR Supermix for Residual DNA Quantification

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 QX200 Droplet Digital PCR System, load
F
20 μl of each reaction mix into a sample well of a DG8Cartridge. Follow subsequent instructions as specified
in the QX200 Droplet Generator Instruction Manual(10031907)
 or the QX200 AutoDG Droplet Digital PCR System,Ffollow instructions in the Automated Droplet GeneratorInstruction Manual (10043138)
 or the QX ONE Droplet Digital PCR System, loadF
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 System
Instruction Manual (10000116512)

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

Time

Numberof Cycles

Hold(QX ONE ddPCR System only)

25

3 min

1

Enzyme activation95

10 min1

Denaturation95

30 sec40

Annealing/extension

60

1 min**

40

Enzyme deactivation98

10 min1

QX200 ddPCR System(optional)

4Infinite

1

QX ONE ddPCR System(required)

251 min1Cycling StepHold* For the C1000 Touch Thermal Cycler, use a heated lid set to 105°C and set
the sample 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:■■

Thermal Cycling Conditions
Follow instructions based on the system in use:
■■

■■

■■

■■

 or the QX200 Droplet Digital PCR System, after dropletF
generation with the QX200 Droplet Generator, carefullytransfer droplets into a clean 96-well plate. Seal the plateusing the PX1 PCR Plate Sealer at 180°C for 5 sec. Proceedto thermal cycling (see Table 2)
 or the QX200 AutoDG Droplet Digital PCR System,Fremove the droplet plate containing ddPCR dropletsfrom the Automated Droplet Generator. Seal the plateusing 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 performedby the system itself. Hence, no additional equipment orsample handling is required for this step. Refer to theQX ONE Droplet Digital PCR System Instruction Manual(10000116512) for plate setup instructions. Use appropriate
thermal cycling conditions as specified in Table 2

 or the QX200 Droplet Digital PCR System and theF
QX200 AutoDG Droplet Digital PCR System, refer tothe QX200 Droplet Reader and QuantaSoft Software
Instruction Manual (10031906)
 the QX ONE Droplet Digital PCR System, refer toFor
the QX ONE Droplet Digital PCR System Instruction
Manual (10000116512) and the QX ONE Software
Instruction Manual 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.

 he concentration reported is copies/µl of the final
T1x ddPCR reaction.

Visit bio-rad.com/web/ddPCRRDQ for more information.
BIO-RAD, DDPCR, DROPLET DIGITAL PCR, and QUANTASOFT are trademarks ofBio-Rad Laboratories, Inc. in certain jurisdictions.
Purchase of Digital PCR products from Bio-Rad Laboratories includes a limited,
non-transferable right under our intellectual property for you to use the products
for your internal research purposes only. Unless we expressly state otherwise inadditional Terms and Conditions, no rights are granted for you to distribute or resell
the products or to use the products for commercial applications, such as contract
services or fee for services. No rights are granted for diagnostic uses. Information
concerning a license for such uses can be obtained from Bio-Rad Laboratories. Itis the responsibility of the purchaser/end user to acquire any additional intellectual
property rights that may be required. The QX200, QX200 AutoDG, and QX ONE
Droplet Digital PCR Systems and/or their use is covered by claims of U.S. patents,and/or pending U.S. and non-U.S. patent applications owned by or under license to
Bio-Rad Laboratories, Inc. See bio-rad.com/en-us/trademarks for details.
All trademarks used herein are the property of their respective owner.

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