PrimeTime® Gene Expression Master Mix
ProductQuantity*

Catalog #1 x 1 mL

1055770

1 x 5 mL

1055772

5 x 5 mL

1055771

20 x 25 mL

1072102

PrimeTime® Gene Expression Master Mix* Separate tubes of reference dye are included with all orders, except for the 20 x 25 mL size (if needed, reference dye can be ordered		 by contacting custcare@idtdna.com).
Go to www.idtdna.com for safety data sheets (SDSs) and certificates of analysis (COAs) for IDT products. Visit www.idtdna.com/protocols toverify that you are using the most current version of this protocol.

Contents and storage conditions

Storage and stability

PrimeTime Gene Expression Master Mix is a ready-to-use,
2X concentrated master mix that is designed for use inprobe-based, real-time quantitative PCR. PrimeTime GeneExpression Master Mix contains an antibody-mediated, hot-start
DNA polymerase, dNTPs, MgCl2, enhancers, and stabilizers.
®

In addition, a reference dye is provided as a separate component,
making this master mix compatible for use on both referencedye–dependent and –independent instrument systems.

–15 to –30°C in a constant temperature (non–frost-free) freezer
until the end of the month indicated in the expiration dateor

2 to 8°C for 1 month
Avoid repeated freezing and thawing
Avoid prolonged exposure of reference dye to light

Prepare reaction mix and run PCR
1. Prepare reagentsa. Thaw the PrimeTime Gene Expression Master Mix, reference dye, primer and probe solutions, and template DNA on ice.
b. Briefly vortex to mix reagents thoroughly.
c. Quickly centrifuge to collect solutions at the bottom of tubes.

2. Add reference dye, as needed
If needed (see Table 1), add the appropriate amount of reference dye to the PrimeTime Gene Expression Master Mix (Table 2).

Reference dye

PCR system

High

7900HT Fast and 7300 Real-Time PCR System (Thermo Fisher Scientific)

X

StepOne™ and StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific)

X

Low

Mx3005P and Mx4000P qPCR System (Agilent)X

7500 Real-Time PCR System (Thermo Fisher Scientific)

X

Viia™7 Real-Time PCR System (Thermo Fisher Scientific)

X

QuantStudio™ Flex Systems (Thermo Fisher Scientific)

X

™

™

None

CFX, iQ , and Opticon Real-Time PCR Detection Systems (Bio-Rad)

XLightCycler® Real-Time PCR Systems (Roche)

X™

™

Table 1. Reference dye concentration levels required by various PCR systems. For instruments not listed, please check with the manufacturer.
Dye volume (µL)
High reference dye systems

Low reference dye systems

To a 1 mL stock vial of master mix, add:

404To a 5 mL stock vial of master mix, add:

200201000

100

To a 25 mL stock vial of master mix, add:

Table 2. Amount of reference dye to add to PrimeTime Gene Expression Master Mix.
®

For Research Use Only.

Version 11072639

3. Prepare the Reaction Mixa. Make enough Reaction Mix for the number of reactions needed.		 Note: Include controls (no reverse transcriptase, if needed; no template control; positive control) and 1–3 additional reactions to		 account for pipetting errors.
b. Combine and thoroughly mix all components (Table 3), except for the DNA template, which will be added separately in Step 4.		Note: Add enough Nuclease-Free Water (Catalog # 11-04-02-01 or 11-05-01-14) so that the total reaction volume at the end of Step 4		 is 20 µL or 10 µL, depending on your chosen reaction volume.

PrimeTime® qPCR Assay reactions(Premixed primers and probe)
Component
PrimeTime® Gene Expression
Master Mix (2X)

PrimeTime qPCR Assay*(20X)®

DNA template(Add in Step 4)

Final
conc. oramount1X

1X

3 pg to100 ng

Nuclease-Free WaterqPCR using separate primers and probe

VolumeVolumeper 20 µL per 10 µL
reactionreaction
10 µL

1 µL

5 µL

Final
conc. oramount

Volumeper 20 µL
reaction

Volumeper 10 µL
reaction

PrimeTime® Gene Expression
Master Mix (2X)

1X

10 µL

5 µL

Forward and reverse primers

250–
1000 nM
each

Variable

Variable

Probe(s)150–250 nM
each

Variable

Variable

3 pg to100 ng

2–5 µL

2–4 µL

Bring to20 µL

Bring to10 µL

Component

0.5 µL

2–5 µL

2–4.5 µL

DNA template(Add in Step 4)

Bring to20 µL

Bring to10 µL

Nuclease-Free Water* To order, visit www.idtdna.com/primetime.

Table 3. Reaction mixes. The volumes provided are “per reaction”. Thus, determine final component volumes by multiplying each volume by
the total number of reactions.
c. Dispense equal aliquots of the Reaction Mix into the wells of a qPCR plate that is compatible with your real-time PCR instrument.

4. Add DNA templatea. Add 3 pg to 100 ng of DNA template or controls to the wells containing Reaction Mix (Table 3).
b. Seal with optically transparent film.
c. Gently agitate to ensure thorough mixing, and centrifuge briefly to remove air bubbles and collect the reaction at the bottom of the wells.

5. Set up cycling program
Program the appropriate PCR cycling protocol on your real-time PCR instrument (Table 4).

StepCycles

Temperature

Fast cycling (min:sec)

Standard cycling (min:sec)

195°C

3:003:00

Denaturation

95°C

0:05

0:15

Annealing/Extension*60°C

0:30

1:00

4°C

Up to 24 hr

Up to 24 hr

Polymerase activation
Amplification

Hold, if needed

35–45

1

* This is a general starting point. The annealing/extension temperature or time may need to be adjusted based on primer sequences. However,		
do not change the polymerase activation conditions.

Table 4. Cycling protocol.

6. Run PCR
a. If the plate was stored before PCR, vortex and then centrifuge briefly.
b. Place the plate in the real-time PCR instrument and start the cycling program.
For additional information or assistance, visit www.idtdna.com/protocols to download the PrimeTime® qPCR Application Guide or email
us at applicationsupport@idtdna.com.
For Research Use Only.
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or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks. PCR-10018-PR 09/15

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