This is 2X concentrated solution for PCR reaction which contains dNTPs, DX/DT HiFi-Pfu DNA Polymerase, Mg+2 and other critical reaction components. It doesn’t contain primers and DNA template. Pfu Mastermix is designed for routine PCR, cloning and other applications which require higher fidelity.
DX/DT High Fidelity Pfu DNA Polymerase catalyzes DNA dependent polymerization from 5’→3’, in presence of Mg2+ and also exhibits 3’→5’ exonuclease activity for proof reading, which enables the polymerase to correct base insertion errors. DX/DT Pfu DNA polymerase is used across various labs in India, especially for cloning and site directed mutagenesis application due its extreme versatile and user friendly compositions. The mastermix comes with pre loaded dye which can be directly loaded on gel post PCR.
Offers exceptional fidelity in DNA amplification, ensuring minimal errors in replicated sequences.
PCR Amplifications upto 12KB is supported.
Suited for site directed mutagenesis with great tolerance.
Suitable for library preparation in next-generation sequencing (NGS) applications.
High tolerance for many PCR inhibitors/reagents.
Comes with preloaded blue dye for direct gel loading.
Can also be used for colony PCR screening tests.
Ultrafast delivery. 2 Hrs for Bangalore and 2-3 working days across India.
Features
Source
The Mastermix contains HiFi DX/DT mutant Pfu DNA Polymerase whose gene was cloned from Pyrococcus furiosus and expressed in E.Coli.
1ng of Lambda (λ) phage DNA was taken for amplification. 500bp , 3kb and 8kb fragments were amplified using HiFi PFU Mastermix. Average GC content: 57%
PCR Conditions
Lambda template (1µL), forward & reverse primers (1µL each), Pfu Mastermix (25µL) and make upto 50µL using Nuclease Free Water.
Cycling conditions and Gel
95℃ (2min), 25 Cycles [ 95℃-30s; 58℃-30s; 72℃-1min/kb], 72℃- 5to 10 minutes. 5µL of sample was loaded directly in 1% Agarose Gel which was run just for 30 minutes.
DX/DT Pfu mastermix has extreme capabilities to detect positive colonies. To demonstrate this, two plasmids were chosen.
Plasmid 1: 6.7 KB total length. Gene fragment: 1.8KB
Plasmid 2: 8KB total length. Gene fragment: 2.9KB
PCR Conditions
One single colony was picked using 10µL tip and suspended in 100µL of nuclease free water. From this suspension, 1µL, 3µL and 1µL(diluted 10 times) were taken for colony PCR.
Forward & Reverse(10µM): 1µL each
Pfu Mastermix: 25µL
Final Volume: 50µL
Cycling conditions and Gel
Plasmid 1: 95℃ (2min), 25 Cycles [ 95℃-30s; 58℃-30s; 72℃-1.5min], 72℃- 2 minutes.
Plasmid 2: 95℃ (2min), 25 Cycles [ 95℃-30s; 58℃-30s; 72℃-3min], 72℃- 5 minutes.
10 µL of sample was loaded directly in 1% Agarose Gel which was run just for 30 minutes.
DX/DT High Fidelity Pfu mastermix is generally used for cloning and site directed mutagenesis. Fidelity and sensitivity is very critical for both step.
Test details
A plasmid was chosen for cloning 2.9kb fragment & was diluted serially; 10ng, 1ng, 100pg and 10pg
PCR Conditions
Sample: Plasmid (8KB total size), target 2.9kb
Cycling conditions and Gel
98℃ (30s), 25 Cycles [ 98℃-10s; 55℃-20s; 72℃-3 min], 72℃- 5 minutes.
10 µL of sample was loaded directly in 1% Agarose Gel which was run just for 30 minutes.
Long time storage: -20℃, 2 years
Day to day usage: 4℃, One Month
Always prevent the master-mix from primer and nucleases contamination.
Protocol for this product can be found here.
All the products are for research purpose only. Not for human diagnostics.
