2×2×96well Microplate Box Type Rotor 

Endonucleases DNA-specific, dsDNase

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Double-Strand Specific dsDNase (dsDNase) is ideal for fast and effective removal of contaminating DNA from PCR master mixes.

Taq polymerases are commonly contaminated by bacterial DNA. This is a problem in PCR based bacterial typing and detection as it might cause false positive results. The unique properties of dsDNase make it suited for removal of contaminating DNA from PCR master mixes prior to addition of DNA template.

In figure 1, a PCR master mix was treated with different amounts of dsDNase before performing a qPCR to measure the contaminating bacterial DNA in the master mix. ArcticZymes dsDNase effectively removed contaminating DNA below known levels of the assay detection limits.

The dsDNase from Arctic shrimp (Pandalus borealis) is recombinantly produced in Pichia pastoris. It cleaves phosphodiester linkages in DNA to yield oligonucleotides with 5’-phosphate and 3’-hydroxyl termini.

The specific activity is estimated to be 30 times higher than that of bovine DNase I. In the presence of magnesium as only divalent cation and using oligos as a substrate, the activity towards dsDNA is 5000-fold higher than towards ssDNA.

The unique double strand-specificity allows specific degradation of dsDNA while leaving shorter ssDNA as primers and probes essentially intact. Easy inactivation by moderate heat (65°C) allows addition of DNA intended for analysis directly after removal of contaminating DNA.

Workflow – Decontamination of PCR master mixes

Recommended applications:

• Decontamination of PCR master mixes
• Removal of genomic DNA from RNA preparations

Key advantages with dsDNase:

• Double-strand DNA specific endonuclease
• High specific activity
• Can be heat-inactivated by moderate heat treatment (65°C for 15 minutes)
• Producing 5′-phospho-oligonucleotide products

Figures

Figure 1. The dsDNase effectively removes contaminated DNA

The dsDNase effectively removes contaminated DNA:

A PCR master mix was preincubated with various concentrations of dsDNase. After treatment, no DNA was amplified in non-template controls.

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Properties

Specificity towards double-stranded DNA

Nucleic acid specificity has been tested towards double- and single-stranded DNA and RNA oligonucleotides. The specificity of dsDNase towards the substrate has been measured using 15-mer oligonucleotides with FAM at 5′ and DarkQuencher® 3′ (Eurogentec). The fluorescence is proportional to enzyme activity. Assay conditions: 25 mM Tris pH 7.5, 5 mM MgCl₂, and 2 μM oligonucleotide.

Relative activities

Substrate                  Relative Activity
dsDNA                           100%
ssDNA                          <0.03%
dsRNA                          <0.01%
ssRNA                           <0.01%

Double-Strand Specific dsDNase (dsDNase) is ideal for fast and effective removal of contaminating DNA from PCR master mixes.

Taq polymerases are commonly contaminated by bacterial DNA. This is a problem in PCR based bacterial typing and detection as it might cause false positive results. The unique properties of dsDNase make it suited for removal of contaminating DNA from PCR master mixes prior to addition of DNA template.

Overview

• Protocol optimized for DNA isolated from a diversity of samples including stool, soil, water, saliva, plant, urine, skin, and more
• Simple and quick workflow: library could be prepared in less than 5 hours
• Component of Norgen’s metagenomics workflow
• A single NGS run can be prepared with up to 384 unique dual-index libraries

Sample type purification kit guide 

The 16S V2-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V2-V3 amplicon libraries to be used for next-generation sequencing on Illumina platforms. All molecular reagents including primers, enzyme mixes, indexes, and buffers are provided. Instructions for PCR clean up with the AMPure XP Magnetic Beads (supplied by customer) are also included for rapid purification of nucleic acid products generated at two steps of the workflow. The library prep workflow could be used for purified DNA inputs from different sources including stool, soil, water, saliva, plant, urine, skin swab, vaginal swab, cheek swab, nasal swab, plasma/serum, tongue swab, gum swab, and others.

The 16S V2-V3 Library Preparation Kit for Illumina has a streamlined procedure that reduces the handling time such that the library prep procedure can be completed in approximately 4 hours (see diagram below). Input DNA is first subjected to targeted PCR to amplify the V2-V3 region of the DNA encoding 16S rRNA. The post-PCR reaction is then cleaned up using AMPure XP beads. Dual index primers are then added using a limited-cycle PCR. The indexed amplicons flanked by 5′ and 3′ barcoded adaptors are then cleaned using AMPure XP beads. The libraries are then ready for quantification, pooling and sequencing.

Details

Supporting Data

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Minimum amount of starting material:	2.5 µL of DNA (5 ng/µL)
Time to complete library preparation:	4 hours

Storage Conditions and Product Stability
Norgen’s 16S V2-V3 Library Prep Kit for Illumina is shipped as one kit box (for the 24 prep kit) or two sub-component kits (for the 96 prep kit). All kits should be stored at -20°C upon arrival.

All kit components should remain stable for at least 1 year when stored at the specified storage conditions.

Step	Component	Cat. 70300 (24 preps)	Cat. 70310, 70320, 70330, 70340 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V2-V3 Primer Mix	70 µL	280 µL
Index PCR (PCR 2)	Indexing Master Mix	660 µL	2 x 1,320 µL
	N7xx Index Primer	50 µL	50 µL
	S5xx Index Primer	70 µL	70 µL
PCR Clean-Up	Resuspension Buffer	2 x 1,250 µL	2 x 5,000 µL
	Nuclease-free water	1,250 µL	1 x 6,000 µL

K-PullG6

SKU: 700004329

100/200 assays per kit

Content:	100 / 200 assays per kit
Shipping Temperature:	Ambient
Storage Temperature:	Short term stability: 2-8oC, Long term stability: See individual component labels
Stability:	> 2 years under recommended storage conditions

Analyte:	Pullulanase/Limit-Dextrinase
Assay Format:	Spectrophotometer
Detection Method:	Absorbance
Wavelength (nm):	400
Signal Response:	Increase
Limit of Detection:	0.18 U/mL for pullulanase preparations (50-fold dilution) 0.01 U/g for limit dextrinase in milled malt
Reproducibility (%):	~ 3%
Total Assay Time:	~ 10 min (Pullanase), ~ 30 min (Limit-Dextrinase)
Application examples:	Assay of microbial pullulanase preparations. Measurement of limit-dextrinase in malt extracts.
Method recognition:	Novel method

PullG6 assay for the measurement of pullulanase employs a water soluble defined substrate, namely 4,6-O-benzylidene-4-nitrophenyl-6³-α-D-maltotriosyl-maltotriose (BPNPG3G3), coupled with the ancillary enzymes α-glucosidase and β-glucosidase. Upon hydrolysis of the substrate at the 1,6-α-linkage by pullulanase or limit-dextrinase, the released 4-nitrophenyl-β-maltotrioside is immediately hydrolysed to glucose and 4-nitrophenol by the concerted action of the α-glucosidase and β-glucosidase enzymes in the reagent mixture. The reaction is terminated and phenolate ions are developed by addition of dilute alkali. The absorbance is read at 400 nm and the value obtained correlates directly with pullulanase activity.

Explore more of our assay kit products for enzyme activity measurement.

Advantages

• High sensitivity 
• Suitable for manual and auto-analyser formats 
• No transglycosylation interference 
• Very cost effective 
• All reagents stable for > 1 year after preparation 
• Very specific
• Simple format
• Standard included