QX200™ ddPCR™ EvaGreen Supermix®

Catalog #Description

1864033
1864034
1864035
1864036

QX200 ddPCR EvaGreen® Supermix, 2 ml (2 x 1 ml vials), 200 x 20 µl reactions
QX200 ddPCR EvaGreen® Supermix, 5 ml (5 x 1 ml vials), 500 x 20 µl reactions
QX200 ddPCR EvaGreen® Supermix, 25 ml (5 x 5 ml vials), 2,500 x 20 µl reactions
QX200 ddPCR EvaGreen® Supermix, 50 ml (10 x 5 ml vials), 5,000 x 20 µl reactions

For research purposes only.

Description
QX200 ddPCR EvaGreen® Supermix is a 2x concentrated,ready-to-use reaction cocktail containing all components —except primers and template — required for Droplet Digital™ PCR(ddPCR). The mixture delivers maximum target specificity andfluorescence amplitude and minimum droplet variability to ensure
precise target quantification. Conventional cycling protocols areused for dye-based detection in Droplet Digital PCR.
The hot-start feature of the polymerase in the supermix
enables partitioning of sample into droplets while keeping
the enzyme inactive at ambient conditions. The supermix has
been optimized to support the amplification and detectionof DNA targets using commercially available EvaGreen®
Assays, and is suitable for use with uracil N-glycosylase(UNG) decontamination protocols. UNG may be purchasedfrom a licensed supplier.
Storage and Stability
QX200 ddPCR EvaGreen® Supermix is stable at –20°C
through the expiration date printed on the label. Once thawed,
it can be stored at 4°C for up to 2 weeks. Repeated freezing
and thawing of the supermix is not recommended.
Quality Control
QX200 ddPCR EvaGreen® Supermix is free of contaminating
DNase and RNase. Stringent specifications are maintained toensure lot-to-lot consistency.
Recommendations for Optimal Results■ Follow general guidelines and recommendations for Droplet
Digital PCR (refer to the Droplet Digital PCR Applications
Guide, bulletin 6407)■

 he concentration of intact human genomic DNA should
Tbe ≤66 ng per 20 μl reaction. If using higher concentrations,digest DNA with a restriction endonuclease that does not
cut target or reference amplicons (see guidelines inDNA Digestion section)

Required Equipment■ Q X200 Droplet Digital PCR System (catalog #1864001),
QX200 AutoDG™ Droplet Digital PCR System (#1864100),
QX600™ Droplet Digital PCR System (#17007769), or
QX600 AutoDG Droplet Digital PCR System (#17008371)■■

 TC Tempo Deepwell Thermal Cycler (#12015392) orP
C1000 Touch Thermal Cycler with 96–Deep Well Reaction
Module (#1851197)
PX1 PCR Plate Sealer (#1814000)

Refer to the QX200 Droplet Reader and QX Manager SoftwareStandard Edition User Guide and QX200 Droplet Generator
Instruction Manual (10000107223 and 10031907, respectively)or the Automated Droplet Generator Instruction Manual(10043138) for ordering information about consumables, suchas oils, cartridges, gaskets, plates, and seals.
Reaction Setup
1.	Thaw all components to room temperature. Mix thoroughly
by vortexing each tube to ensure homogeneity because
a concentration gradient may form during –20°C storage.
Centrifuge briefly to collect contents at the bottom of the tubes.
2.	Prepare samples at the desired concentration before setting
up the reaction mix.
3.	Prepare the reaction mix for the number of reactions needed
according to the guidelines in Table 1. Assemble all requiredcomponents except the sample, dispense equal aliquots
into each reaction tube, and add the sample to eachreaction tube as the final step.
Table 1. Preparation of the reaction mix.
Volume perReaction, µl

Final Concentration

101x

Forward primer

Variable

100–250 nM

Reverse primer

Variable

100–250 nM

Diluted restriction enzyme(see DNA Digestion section)

1

Variable

DNA template* andRNase-/DNase-free water

Variable

Up to 100 ng**

20—

Component
2x QX200 ddPCR
EvaGreen® Supermix

Total volume* Suggested amplicon length: 80–250 bp.
** S ample concentrations >66 ng per reaction and certain applications
may require restriction digestion for optimal target detection. If digestion
is not required, prepare the ddPCR reaction mix without the diluted
restriction enzyme.

4.	Mix thoroughly by vortexing the tubes. Centrifuge briefly toensure that all components are at the bottom of the reaction
tubes. Allow reaction tubes to equilibrate at room temperature
for about 3 minutes.
5.	Once the reaction mixtures are ready, load 20 µl of eachreaction mix into a sample well of a DG8 Cartridge for QX200
Droplet Generator (#1864008) followed by 70 µl of QX200
Droplet Generation Oil for EvaGreen® (#1864005 or 1864006)
into the oil wells, according to the QX200 Droplet Generator

QX200 ddPCR EvaGreen® Supermix

Instruction Manual (10031907). For the Automated Droplet
Generator, follow instructions in the Automated Droplet
Generator Instruction Manual (10043138).
DNA Digestion (recommended)
DNA fragmentation by restriction digestion prior to droplet
generation enables optimal accuracy by separating tandem
gene copies, reducing sample viscosity, and improving
template accessibility for input samples between 66 and 100 ng
per well. Restriction digestion improves overall performance ofDroplet Digital PCR across applications. Choose a restriction
endonuclease that does not cut either the target or reference
amplicon and that is insensitive to methylation. Four-basecutters and high-fidelity enzymes are preferred.
Two strategies may be used to perform restriction digestion ofDNA samples: digestion directly in the ddPCR reaction during
setup or conventional digestion prior to Droplet Digital PCR.
Digestion in ddPCR Reaction
■

■

■

■

■

 fficient digestion of sample DNA can be achieved by direct
Eaddition of restriction enzymes to the ddPCR reaction ilute the restriction enzyme using the recommended
Ddiluent buffer according to the manufacturer’s instructions
and add 1 µl to the ddPCR reaction as outlined in Table 1
 pproximately 2– 5 units of restriction enzyme per 20 µl
AddPCR reaction are recommended
 he addition of restriction enzyme buffers with high salt canTinhibit Droplet Digital PCR and should be avoided
 aeIII, MseI, AluI, HindIII, and CviQI have been observed toHwork well for digestions in ddPCR reactions

Digestion Prior to Droplet Digital PCR
■■■■

■■■■

 estriction enzyme digestion can be carried out asRa separate reaction before ddPCR reaction setup

2.	Proceed to thermal cycling (see protocol in Table 2) andsubsequent reading of droplets in the QX200 Droplet Reader.
Table 2. Thermal cycling conditions.*Cycling Step

Temperature, ºCTime

Ramp Rate

Numberof Cycles

Enzyme activation

95

5 min

1

Denaturation95

30 sec40

Annealing/extension

60

1 min

45 min

90

5 min

1

430 min

1Signal stabilization
Hold402°C/sec

1* 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
the sample volume to 40 µl.

Data Acquisition and Analysis
1. A
 fter thermal cycling, place the sealed 96-well plate in the
QX600 or QX200 Droplet Reader.
2. O pen QX Manager Software to set up a new plate layout
according to the experimental design. Refer to the QX200
Droplet Reader and QX Manager Software Standard Edition
User Guide (10000107223).
3. Under Setup, double click on a well in the plate layout toopen the Well Editor dialog box.
4. D
 esignate the sample name, experiment type, QX200
ddPCR EvaGreen Supermix as the supermix type, target
name, and target type: Ch1 for FAM.
5. Select Apply to load the wells and when finished, select OK.
6. Once the plate layout is complete, select Run to begin thedroplet reading process. Select EvaGreen as the dye set used
and run options when prompted.

 se 5–10 enzyme units per microgram DNA andU
10–20 enzyme units per microgram genomic DNA

7. After data acquisition, select samples in the well selector
under Analyze. Examine the automatic thresholding applied
to the 1-D amplitude data and, if necessary, set thresholds
or clusters manually.

Incubate the reaction for 1 hr at the temperature
recommended for the restriction enzyme

8. T
 he concentration reported is copies/µl of the final
1x ddPCR reaction.

 eat inactivation is not required, but can be considered if
Hlong-term storage is required; do not heat inactivate above
65°C

Visit bio-rad.com/ddPCRsmxEvaG for more information.

DNA purification is not necessary after restriction digestion se a minimum 10-fold dilution of the digest to reduce theUsalt content of the sample in the ddPCR reaction
Store digested DNA at –20°C or below
 repare the ddPCR reaction mix, as outlined in Table 1,
P
without the diluted restriction enzyme

Thermal Cycling Conditions
1. A
 fter droplet generation with the QX200 Droplet Generator,
carefully transfer droplets into a clean 96-well plate, orremove the droplet plate containing ddPCR droplets from theAutomated Droplet Generator. Seal the plate with the PX1PCR Plate Sealer.
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Drive, Hercules, CA 94547
510-741-1000

BIO-RAD, AUTODG, DDPCR, DROPLET DIGITAL, QX200, and QX600 are trademarksof Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are
the property of their respective owner. © 2023 Bio-Rad Laboratories, Inc.
EvaGreen is a trademark of Biotium, Inc. Bio-Rad Laboratories, Inc. is licensed byBiotium, Inc. to sell reagents containing EvaGreen Dye for use in real-time PCR, forresearch purposes only.
Purchase of Digital PCR and/or Single-Cell NGS Sample Preparation products (the“Products”) from Bio-Rad Laboratories is subject to Bio-Rad Laboratories, Inc.
Standard Terms and Conditions of Sale, which can be accessed at https://www.
bio-rad.com/en-us/terms-conditions. Unless we expressly state otherwise inadditional Terms and Conditions, no rights are granted for you to distribute orresell the Products. Unless we expressly state otherwise in additional Terms andConditions, ​no rights are granted for the development or commercialization ofdiagnostic assays for use with the Products without a license from Bio-Rad. It is theuser’s obligation to obtain a commercial license from Bio-Rad for (i) all commercial
uses (not just diagnostic uses) and (ii) sale of assays for use on Bio-Rad’s dPCR andddSEQ instruments. The Products and/or their use are covered by U.S.​and foreign
patents and/or pending patent applications owned by or under license to Bio-Rad
Laboratories, Inc. See https:// www.bio-rad.com/en-us/trademarks.

10000162845 Ver B (10028376)

23-03570423