HiFi Hotstart PCR Mix - BioReagent,DNase, RNase free,PCR Reagent,UltraBio™,Suitable for molecular biology,for DNA and RNA applications,25μL/T

Cat. No.: FP1508888

GRADE & PURITY Suitable for molecular biology, BioReagent, DNase, RNase free, PCR Reagent, UltraBio™, for DNA and RNA applications, 25μL/T

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Application

PCR

Overview

  The HiFi Hotstart PCR Mix features an engineered HiFi Seq Hotstart DNA polymerase that significantly enhances DNA template affinity without requiring auxiliary proteins or additional DNA-binding domains. Compared to wild-type B-family DNA polymerases, this enzyme demonstrates:
✔ Superior processivity: Higher amplicon yield, faster amplification speed, and enhanced sensitivity
✔ Robust performance: Efficient amplification of long fragments (≥15 kb) and complex templates (e.g., high-AT/GC content)
✔ Proofreading activity: Powerful 3’→5’ exonuclease function (error rate 100× lower than wild-type Taq, 10× lower than other B-family polymerases)
✔ Minimal side activity: 5’→3’ polymerase activity with no 5’→3’ exonuclease activity

The kit provides an optimized system for sensitive PCR amplification (down to 1 ng template) and includes:

• HiFi Seq Hotstart DNA polymerase (1 U/50 μL)

• Pre-optimized buffer

• dNTPs

• MgCl₂

Simply add primers and template for high-fidelity amplification.

Catalog	Component	24T	96T	Storage
FP1508888	HiFi Hotstart PCR Mix	600 μL	4×600 μL	-20℃

Applications:

• NGS DNA library enrichment

• Gene expression cloning

• Site-directed mutagenesis

• Intracellular gene mutation analysis

User-Supplied Materials

• Instruments: PCR plates, thermal cycler, vortex mixer, microcentrifuge

• Reagents: Primer pair (5 μM each), nuclease-free water

• Consumables: Pipette tips, 0.2 mL PCR tubes

Protocol

I. Reaction Setup

1. Thaw HiFi Hotstart PCR Mix on ice. Vortex thoroughly if precipitates are visible.

2. Prepare the reaction mix on ice as follows:

Component	Volume per 50 μL Reaction
HiFi Hotstart PCR Mix	25 μl
Primer Pair (each 5 μM)	2~5 μl
DNA Template	x
Nuclease-free water	Up to 50 μl

Notes:

1. Total volume: 10–50 μL (scale mix proportionally; >50 μL not recommended).

2. Default concentrations: 2.5 mM Mg²⁺, 0.2 mM dNTPs, 1 U polymerase. Adjust Mg²⁺/dNTPs if needed.

3. For initial testing: Use 10–100 ng genomic DNA per 50 μL reaction.

      3. Mix gently by pipetting; centrifuge briefly.

II. Thermal Cycling

Optimization Notes:
* Pre-denaturation:

• Standard: 3 min at 95°C

• For GC-rich templates (≥70% GC): Extend to 5 min or use 98°C.

** Annealing Temperature:

• Start at 56°C (higher than conventional PCR due to optimized buffer).

• Adjust in 3°C increments if amplification fails.

*** Extension Time:

• ≤1 kb: 15 sec/kb

• >1 kb or higher yield needed: 30–60 sec/kb

• Two-step PCR: Combine annealing/extension at 65–68°C.

**** Cycle Number:

• ≤25 cycles recommended for highest fidelity (more cycles increase error rates).

Specifications

Specifications & Purity

BioReagent, DNase, RNase free, PCR Reagent, UltraBio™, Suitable for molecular biology, for DNA and RNA applications, 25μL/T

Stability And Storage

Store at -20℃ long term (12 months). Avoid freeze/thaw cycle. Upon receipt, it is recommended to aliquot.

Storage

Store at -20°C, Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent, DNase, RNase free, for DNA and RNA applications, PCR Reagent, Suitable for molecular biology, UltraBio™