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Description


Chai’s Dry PCR Master Mix is an optimized for routine PCR and qPCR. It is lyophilized for ambient temperature shipping and can easily be reconstituted with the provided buffer. The mix is optimized for Real-Time PCR, capable of 100% PCR efficiency, and is compatible with all standard probe and dye-based chemistries. The mix combines numerous PCR enhancers and stabilizers with a novel aptamer-based hot start mechanism, resulting in exceptional qPCR performance. This mix is validated for products between 100 bp and 5 kb.

Pcr master mix dry hot start manual cover

Specifications


Concentration (Reconstituted)2X
Shipping ConditionsAmbient Temp
Storage Temperature-20 – 4 °C
Storage Temperature (Reconstituted)-20 °C
Exonuclease Activity5' → 3'
Hot StartYes
Recommended Reaction Size10 – 50 µL

Composition


ComponentAmount
Taq polymerase50 U/mL
Aptamer20 nM
KCl100 mM
MgCl26 mM
TrisCl pH 8.620 mM
Glycerol10%
Trehalose200 mM
BSA0.4 mg/mL
Detergents0.26%
dNTPs (each)600 µM

Performance


Amplification of Ara h2 Gene Target Using Chai PCR Master Mix + Hot Start (DRY)
Amplification of Arah2 Gene Target Using Chai PCR Master Mix + Hot Start (DRY)
Five ten-fold serial dilutions of Ara h2 gene fragments were used as template to amplify a 124 bp product.

Ara h2 Standard Curve Amplified With Chai PCR Master Mix + Hot Start (DRY)
GAPDH Standard Curve Amplified With Chai PCR Master Mix + Hot Start 2X
Five ten-fold serial dilutions of Ara h2 gene fragments were used to create a PCR standard curve. A 124 bp product was amplified using Chai PCR Master Mix + Hot Start 2X (Dry) and Chai Green dye, with an efficiency of 93.76%, slope of -3.481 and R2 value of 0.99997.

Duplex TaqMan Assay Using Chai PCR Master Mix + Hot Start (DRY)
Duplex TaqMan Assay Using Chai PCR Master Mix + Hot Start 2X
A GAPDH-HPRT gene fragment was used as template. A 123 bp GAPDH amplicon was detected using a FAM labeled TaqMan Probe, and a 98 bp HPRT amplicon was detected using a HEX labeled TaqMan probe. Amplification performed with Chai PCR Master Mix + Hot Start (DRY).

Protocol


Reconstitution: Remove seal and cap from the vial of freeze-dried PCR Master Mix with Hot Start. Add 0.66 mL reconstitution buffer to the freeze-dried vial. Gently pipette up and down at least 10 times to ensure solution homogeneity. Avoid bubbles. Do not vortex. The liquid 2X Master Mix is now ready for use. 


Reaction Set Up:

Assemble the reaction components on ice (preferred) or at room temperature (up to 30 °C). The recommended reaction volume is 25 μL. Reaction volumes between 10 and 50 μL may be used; scale up/down the reaction components accordingly. Transfer the reaction(s) to a thermocycler. No separate activation step is required. Final concentration of the Master Mix in the reaction should be 1X.


Component25 µL REACTION VOLUME
Forward Primer0.2 – 1 μM
Reverse Primer0.2 – 1 μM
Template DNA1 ng – 1 µg Genomic
0.5 pg – 5 ng Plasmid / Viral
1 ng – 100 ng cDNA
qPCR Detection1.25 μL 20X Chai Green
50-250 nM Probe
2X Master Mix with Hot Start12.5 µL
Nuclease-Free WaterBring up to 25 µL

Thermocycling Conditions


Three-Step PCR
StepTempTime
Initial denaturation95 °C2 min
 Denature95 °C15 – 30 s
 Anneal45 – 68 °C15 – 30 s
 Extend68 °C1 min/kb
Final Extension68 °C5 min
Hold4 °C

  Cycle 30 – 40x

Two-Step PCR

When primer annealing temperatures are above 60 °C, a two-step PCR can be used.

StepTempTime
Initial denaturation95 °C2 min
 Denature95 °C15 – 30 s
 Anneal/Extension60 °C1 min/kb
Final Extension68 °C5 min
Hold4 °C

  Cycle 30 – 40x

Downloads


Product Manual Download
Safety Data Sheet (SDS) Download

Certificate of Analysis


Lot 1991225 Download
Lot 6333575 Download