Overview

• Convenient ready-to-use solution
• High sensitivity and yield
• Robust amplification
• Available in 3 convenient sizes: 100, 200 and 500 reactions (20 µL vol)
• This product is also a recommended add-on to be used with Norgen’s COVID-19 Primer and Probe Solutions and COVID-19 Positive Control 

Norgen’s 2X One-Step RT-PCR Master Mix is a ready-to-use solution that contains components required for RT-PCR amplification of RNA templates. The mix includes M-MuLV reverse transcriptase, Taq DNA polymerase, dNTPs, reaction buffer, MgCl₂, KCl, and a PCR enhancer/stabilizer. The user needs only to add the template, the primer set and water to the Master Mix to set up the RT-PCR reaction. This convenient 2X One-Step RT-PCR Master Mix reduces the time required to set up PCR reactions and reduces the possibility of contamination, particularly when preparing large numbers of reactions. The optimized master mix allows for robust amplification of RNA templates with high yields of PCR products.

Taq DNA Polymerase is a highly thermostable DNA polymerase that possesses a 5´→ 3´ polymerase activity and a very low 5´→ 3´ exonuclease activity. The source of Taq included with Norgen’s 2X One-Step RT-PCR Master Mix is an E. coli strain with a cloned Taq DNA Polymerase gene from Thermus aquaticus YT-1. M-MuLV Reverse Transcriptase is an RNA-directed DNA polymerase that can synthesize a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template. The source of the Reverse Transcriptase included with Norgen’s 2X One-Step RT-PCR Master Mix is an E. coli strain with a cloned reverse transcriptase gene from M-MuLV.

Norgen’s 2X One-Step RT-PCR Master Mix is available in 3 convenient sizes:

Cat # 28113 – 100 reactions (sufficient for 100 reactions x 20 µL reaction volume)
Cat # 28114 – 200 reactions (sufficient for 200 reactions x 20 µL reaction volume)
Cat # 28115 – 500 reactions (sufficient for 500 reactions x 20 µL reaction volume)

Details

Supporting Data

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Reagents supplied – Cat # 28114

• 2X One-Step RT-PCR Master Mix (2 Vials, 100 Reactions each) – Sufficient reagent for 200 x 20 µL reactions

Storage Conditions and Product Stability
2X One-Step RT-PCR Master Mix should be stored at -20°C. For everyday use an aliquot can be stored at 4°C for up to 3 months. Repeated freeze-thaw cycles are not recommended. When stored at the proper temperature this reagent is stable for at least 1 year.

Introduction

Hipure Microbial RNA kit is suitable for extracting high-purity total RNA from bacterial/yeast culture medium. This kit combines high-efficiency Magzol Reagent (one-step extraction reagent) and silicagel column purification technology to complete the extraction of high-purity total RNA in only 40 minutes. The obtained RNA can be directly used in RT-PCR, Northern blot, poly-A + purification, nucleic acid protection and in vitro translation.

Details

Specifications

Features	Specifications
Main Functions	Isolation total RNA from bacteria, yeast cells
Applications	RT-PCR, Northern blot, poly-A + purification, nucleic acid protection and in vitro translation
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Bacteria, yeast cells
Sample amount	Bacteria: <10^9；Yeast cells：<2 x10^7
Elution volume	≥30μl
Time per run	≤40 minutes
Liquid carrying volume per column	800µl
Binding yield of column	100µg

Principle

Hipure silica gel column is based on glass fiber filter membrane with high binding force. Under the condition of high concentration of ionizing agent (such as guanidine hydrochloride or guanidine isothiocyanate), the filter membrane can adsorb nucleic acid through hydrogen bond and electrostatic, while protein and other impurities are not adsorbed and removed. The filter membrane adsorbed with nucleic acid is washed to remove the residual protein and salt. Finally, the nucleic acid adsorbed on the filter membrane can be washed out with low salt buffer (such as buffer TE) or water. The obtained nucleic acid has high purity and can be directly used in various downstream experiments.

Advantages

• Fast – several samples can be extracted in 30 minutes
• High purity – the purified RNA can be directly used in various downstream applications
• High recovery – RNA can be recovered at the level of PG
• Good repeatability – silica gel column purification technology can obtain ideal results every time
• Broad spectrum – it can deal with various bacteria, including Gram-positive bacteria that are difficult to be lysed
• Sufficient components – the kit contains carried wall breaking enzymes and glass beads

Kit Contents

Contents	R418202	R418203
Purification Times	50 Preps	250 Preps
HiPure RNA Mini Columns	50	250
2ml Collection Tubes	50	250
Glass Beads (0.1-0.6mm)	30 g	150 g
DNase I	600 μl	5 x 600 μl
DNase Buffer	6 ml	30 ml
Protease Dissolve Buffer	1.8 ml	15 ml
Buffer ATL	50 ml	200 ml
Buffer PHC	50 ml	200 ml
Buffer GXP2*	20 ml	100 ml
Buffer RW1	50 ml	250 ml
Buffer RW2*	20 ml	2 x 50 ml
RNase Free Water	10 ml	30 ml

Storage and Stability

Buffer PHC should be stored at 2–8°C upon arrival. DNase I should be stored at -20°C. However, short-term storage (DNase I up to 1 weeks, Buffer PHC up to 12 weeks) at room temperature(15–25°C) does not affect their performance. The remaining kit components can be stored at roomtemperature (15–25°C) and are stable for at least 18 months under these conditions.

Hipure Microbial RNA kit is suitable for extracting high-purity total RNA from bacterial/yeast culture medium. This kit combines high-efficiency Magzol Reagent (one-step extraction reagent) and silicagel column purification technology to complete the extraction of high-purity total RNA in only 40 minutes. The obtained RNA can be directly used in RT-PCR, Northern blot, poly-A + purification, nucleic acid protection and in vitro translation.