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Cod Uracil-DNA Glycosylase (Cod UNG) from Atlantic Cod is the only commercially available UNG enzyme that is completely and irreversibly inactivated by moderate heat treatment. The enzyme is produced in a recombinant E. coli (ung-) strain that contains a modified Cod UNG gene.

Key Features:

• Heat-labile – Completely and irreversibly inactivated at 55°C
• Contamination control – ideal in applications below
• Use of Cod UNG makes contamination control possible in RT-PCR
• Does not degrade PCR product post-PCR. This makes downstream use of the PCR product possible
• High purity enzyme, tested free of contaminating nucleases
• Detergent free
• Post-PCR Analysis – Enables post-PCR analysis

Applications

Ideal for contamination control in 

• PCR carry-over prevention
• RT-PCR
• RT-qPCR
• qPCR
• kPCR 
• RT-LAMP

Heat-labile Uracil-DNA Glycosylase

There are several commercially available Uracil-DNA glycosylases on the market today. Most of them are of bacterial origin and work well if you have no intention to further analyze the PCR products post-PCR. However, if you want to store your PCR products for downstream analysis such as cloning and sequencing, the reactivation of UNG and subsequent degradation of your PCR products are a problem with most of the commercially available UNGs. Cod UNG from ArcticZymes is completely and irreversibly inactivated by heat thus ensuring that sample integrity is maintained long-term regardless of storage conditions.

This is illustrated in figure 1, below

Figures

Properties

Recommended Protocols

1. Contamination control in PCR, qPCR and one-step RT-qPCR

Cod UNG works in all commercially available master mixes. 

Be sure that you have used dUTP containing dNTP mixes in your previous PCR experiments.

• Add 0.2 U Cod UNG directly to your 20 µl PCR reaction.
• pre-incubate for 5 min at room temperature.
• For RT-qPCR, reverse transcribe your RNA at 50-55°C.
• Run your PCR.
• Store your PCR product at -20°C or 4°C degrees.

2. Contamination control in RT-LAMP

Cod UNG is ideal for contamination control in RT-LAMP.  
One unit of Cod UNG per 30 μl reaction is sufficient for removing even high concentrations of carry-over contamination.

• Ensure that you use dNTP mixes containing dUTP in your experiments.
• Check that the RT-LAMP reaction is compatible with dUTP by running side-by-side reactions containing different ratios of dUTP to dUTP (100% dUTP, 90% dUTP, 80% dUTP and 0% dUTP).
• Add 1 U Cod UNG directly to your 30 µl RT-LAMP reaction.
• Prepare the reaction mix on ice.
• Analyze your RNA at 65°C, no preincubation is necessary.

Please refer to Protocol for Carry-over Contamination Control

Ordering Info

Cod Uracil-DNA Glycosylase (Cod UNG) from Atlantic Cod is the only commercially available UNG enzyme that is completely and irreversibly inactivated by moderate heat treatment. The enzyme is produced in a recombinant E. coli (ung-) strain that contains a modified Cod UNG gene.

Introduction

HiPure Gel Pure DNA Kit uses proprietary chemistry and HiPure technology to recover DNA fragments between 60bp-10kbp with yields exceeding 80%. DNA is suitable for ligations, PCR, sequencing, restriction digestion, or various labeling reactions. In addition, this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions.

Details

Specifications

Principle

The HiPure system uses a simple bind-wash-elute procedure. Gel slices are dissolved in a buffer containing a pH indicator, allowing easy determination of the optimal pH for DNA binding, and the mixture is applied to the column. Nucleic acids adsorb to the silica-gel membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in subsequent applications.

Advantages

• High recovery efficiency – ≥80% DNA recovery
• General – recover DNA from gel or enzyme-driven reaction solutions such as PCR
• Fast – isolation can be completed in 10-15 minutes by column gel method
• Great cost-effectiveness performance

Kit Contents

Storage and Stability

The Kit should be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions. If any precipitates form in the buffers, warm at 37℃ to dissolve.

Experiment Data

HiPure Gel Pure DNA Kit uses proprietary chemistry and HiPure technology to recover DNA fragments between 60bp-10kbp with yields exceeding 80%. DNA is suitable for ligations, PCR, sequencing, restriction digestion, or various labeling reactions. In addition, this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions.

cfDNA Purification Kit (Magnetic Beads)

The cfDNA Purification Kit (Magnetic Beads) was developed for cell free DNA (cfDNA) enrichment by separating genomic DNA contamination from isolated cfDNA samples.

Many diagnostic technologies for detection of disease signals in cfDNA begin with isolation and purification of DNA from liquid biopsy that include urine, plasma, cerebrospinal fluid. The most widely explored biotechnology is assays used to detect cancer-derived plasma cfDNA. Silica-based magnetic bead cfDNA isolation kits can reliably extract total DNA from plasma, but typically yield a large variation in cfDNA that includes the presence of genomic DNA that often depends on tumor stage, tumor size, or healthy status individuals. Most of the commercial cfDNA isolation kits can’t specifically recover the cfDNA while leaving the high molecular weight genomic DNA behind. The presence of genomic DNA can lead to decreased sensitivity or inconsistent results in downstream applications such as next-generation sequencing (NGS), PCR, QPCR, and digital PCR etc.

Therefore, an additional purification step to enrich cfDNA before downstream methods helps to improve signal from fragments that originate from cancer cells. A proportion of cancer-derived cfDNA fragment signals are below 100 bp and are often not detectable except by qPCR or single-stranded DNA based library preparation for NGS (1, 2, 3). Furthermore only 1% of cancer-derived fragments are found above 400 bp (1, 2). Capture of size-selected fragments between 90-150 bp improved detection of cancer by 2-4 fold (4). Furthermore, TF-bound and protected cfDNA fragments are also being investigated for active cancer-specific signals down to 35-80 bp (5, 6).

This kit uses Dual Solid Phase Reversible Immobilization (SPRI) technology for cfDNA purification. Most Dual SPRI procedures do NOT recover fragments below 100 bp. The kit can be used for the enrichment of cfDNA isolated from liquid biopsies, plasma, serum, and urine. The kit separates cfDNA (50-500 bp) and genomic DNA, and recovers of 90% of the cfDNA without the high molecular weight genomic DNA with high efficiency. Fragments at 500 bp and above may also be retained. Both the 50-500 bp and >500 bp DNA fractions can be used for downstream applications such as single-stranded or double stranded NGS library prep, qPCR, ddPCR, and other methods.

Features

• Separation of cfDNA and genomic DNA; Recovery of both types of DNA
• Recovery of cfDNA (50-500 bp)
  • As short as 50 bp can be recovered
• Recovery of high molecular weight genomic DNA
• Removal of unwanted components and other impurities
• Automation friendly

Examples of cfDNA purification. Both cfDNA and genomic DNA can be recovered separately.

The range of recovered small DNA fragments is from 50 to 500 bp. The input DNA are mixtures of sheared small DNA fragments and intact genomic DNA. The ratios of sheared DNA fragments versus genomic DNA are indicated.

Recovery rates of cfDNA and genomic DNA.

Many diagnostic technologies for detection of disease signals in cfDNA begin with isolation and purification of DNA from liquid biopsy that include urine, plasma, cerebrospinal fluid. The most widely explored biotechnology is assays used to detect cancer-derived plasma cfDNA. Silica-based magnetic bead cfDNA isolation kits can reliably extract total DNA from plasma, but typically yield a large variation in cfDNA that includes the presence of genomic DNA that often depends on tumor stage, tumor size, or healthy status individuals. Most of the commercial cfDNA isolation kits can’t specifically recover the cfDNA while leaving the high molecular weight genomic DNA behind. The presence of genomic DNA can lead to decreased sensitivity or inconsistent results in downstream applications such as next-generation sequencing (NGS), PCR, QPCR, and digital PCR etc.