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Description


Chai’s 2X PCR Master Mix with Hot Start is an optimized ready-to-use solution for demanding applications. The mix is optimized for Real-Time PCR, is 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 hot start manual cover @2x

Specifications


Concentration2X
Storage Temperature-20 °C
Shipping ConditionsIce
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


Lambda Phage Amplification Using Chai PCR Master Mix + Hot Start 2X
Lambda Phage Amplification Plot Using Chai PCR Master Mix + Hot Start 2X
Amplification plot of lambda phage amplification using Chai PCR Master Mix + Hot Start 2X. Ten-fold serial dilutions of lambda phage DNA were used at template to amplify a 500 bp product.

GAPDH Standard Curve Amplified With Chai PCR Master Mix + Hot Start 2X
GAPDH Standard Curve Amplified With Chai PCR Master Mix + Hot Start 2X
Five ten-fold serial dilutions of a GAPDH gene fragment were used to create a PCR standard curve. A 123 bp product was amplified using Chai PCR Master Mix + Hot Start 2X and Chai Green dye, with an efficiency of 101%, slope of -3.328 and R2 value of 1.000.

Duplex TaqMan Assay Using Chai PCR Master Mix + Hot Start 2X
Duplex TaqMan Assay Using Chai PCR Master Mix + Hot Start 2X
Ten-fold serial dilutions of a GAPDH-HPRT gene fragment were 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 2X.

Protocol


Thaw the 2X PCR Master Mix with Hot Start at room temperature.Spin the Master Mix briefly in a microcentrifuge to collect the material in the bottom of the tube.


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
2X Master Mix with Hot Start12.5 µL
Nuclease-Free WaterBring up to 25 µL

THERMOCYCLING CONDITIONS

Three-Step PCR
StepTempTime
Initial denaturation95 °C30 s - 2 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 °C30 s - 2 min
 Denature95 °C15 - 30 s
 Anneal/Extension45 - 68 °C1 min/kb
Final Extension68 °C5 min
Hold4 °C

  Cycle 30 - 40x

Downloads


Product Manual Download
Safety Data Sheet (SDS) Download