CHAI Sahara Hot Start PCR Master Mix

by CHAI
Rs. 15,984.00
SKU R02151S

CHAI Hot Start PCR Master Mix is optimized for quantitative Real-Time PCR applications. Aptamer-based hot start technology prevents non-specific amplification at lower temperatures Robust mix for a variety of template types and product lengths.

Make: CHAI

Catalog No: R02151S

Description:

CHAI Hot Start PCR Master Mix 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.

CHAI Hot Start PCR Mix Features:

  • Outstanding thermal stability. Can stay at 25 ºC up to three months without losing the stability
  • Reduced non-specific amplification and primer dimers due to the Aptamer-based hot start system
  • Amplifies GC-rich targets with up to 70% GC-content and up to 4 kb from genomic DNA
  • Gives clear bands
  • High efficiency
  • Fast and effective hot-start

Specifications:

Concentration 2X
Shipping Conditions Ice
Storage Temperature -20 °C
Exonuclease Activity 5' → 3'
Hot Start Yes
Recommended Reaction Size 10 – 50 µL

 

CHAI Hot Start PCR Master Mix includes:

COMPONENT AMOUNT FUNCTION
Taq polymerase 50 U/mL Extends the DNA strand
Aptamer 20 nM Inhibits Taq activity at low temp
KCl 100 mM Stabilizes the DNA strands
MgCl2 6 mM Co-factor for Taq polymerase
TrisCl pH 8.6 20 mM Buffer
Glycerol 10% Enhancer and stabilizer
Trehalose 200 mM Enhancer
BSA 0.4 mg/mL Enhancer and stabilizer
Detergents 0.26% Stabilizer
dNTPs (each) 600 µM Required for strand elongation

 

Protocol:
Thaw the 2X PCR Master Mix with Hot Start to bring it to room temperature. Collect the material at the bottom of the tube after spinning the Master Mix briefly
in a microcentrifuge.

Reaction Set-Up for CHAI Hot Start PCR :

Keep the reaction components on ice. 25 µL is the recommended volume for the reaction. You can use reaction volume between 10 to 50 µL also. Mix the components accordingly.
Reactions can be transferred to a thermocycler. Master Mix concentration in the final reaction mixture should be 1X.

COMPONENT 25 µL REACTION VOLUME
Template DNA 0.2 - 1 µM
Forward Primer 0.2 - 1 µM
Reverse Primer

1 ng - 1 µg Genomic

0.5 pg - 5 ng Plasmid / Viral

1 - 100 ng cDNA

qPCR Detection

1.25 µL 20X Chai Green

50 - 250 nM Probe

2X Master Mix 12.5 µL
Nuclease-Free Water Bring up to 25 µL

 

CHAI Hot Start PCR: Thermocycling Conditions

Three-Step PCR

STEP Temperature TIME
Initial denaturation 95 °C 30 s - 2 min
Denature 95°C 15 - 30 s
Anneal 45 - 68 °C 15 - 30 s
Extend 68°C 1 min/kb
Final Extension 68°C 5 min
Hold 4°C  

Cycle 30 - 40x

Two-Step PCR

STEP Temperature TIME
Initial denaturation 95 °C 2 min
Denature 95°C 15 - 30 s
Anneal /Extention 60 °C 15 - 30 s
Final Extension 68°C 5 min
Hold 4°C  

Cycle 30 - 40x

CHAI Hot Start PCR: Shipping, Storage, and Stability
High-temperature Stability 8 days @ 50 ºC, 3 months @ 25 ºC
Freeze/Thaw Cycles Up to 20
Shipping Conditions Ambient
Storage Conditions -20 ºC, or 4 ºC for 6 months. Protect from light

 

Performance:

High Efficiency and Linearity
 
Ara h2 gene fragment       E = 98.2%, R2 = 1.000
 

CHAI Hot Start PCR

Fast and Effective Hot start

When salmon sperm assay with 67% GC content was run with and without the Sahara Hot Start aptamer, the aptamer hot start successfully prevents the non-specific products seen in the non-hot start assay.

CHAI Hot Start PCR

What is Hot Start PCR?

Hot start PCR is a technique used to reduce unwanted amplification of non-specific DNA fragments. The polymerases in Hot start PCR mix are thermally stable even at room temperature. At this temperature, polymerase activity is inhibited by aptamer technology, antibody interaction or by chemical modification. When the optimum temperature is reached during the polymerase reaction, the inhibitor dissociates from the polymerase kick-starting the polymerase reaction.

General Guidelines for PCR

Primers & Probes: Numerous cost-free software can be spotted online to design or evaluate primers for PCR as well as qPCR. Primers & probes are generally 18 to 30
nucleotides long with a GC concentration of 40 to 60%. Final concentrations of both the primers have to be optimized between 0.1 µM to 0.5 µM. The optimal concentration is one that gives the least Cq value. Optimize probe concentration to a test range of 0.1 µM and 0.5 µM. The best probe concentration is the one that gives the least Cq value as well as good fluorescence
Ingredients: Magnesium is a cofactor necessary for Taq polymerase activity. The magnesium ion concentration in the 1X buffer is 3 mM. Extra magnesium can be included if more optimization is needed. The amplification of GC abundant design templates may be enhanced by utilizing enhancers like DMSO or formamide to stabilize the template for amplification.

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