resDNASEQ E.coli Residual DNA Quantitation kit (Without DNA Control)

Propargyl-PEG2-NHS ester is a PEG linker with a propargyl group that can participate in copper catalyzed azide-alkyne Click Chemistry and NHS ester that can be used to label amine-containing entitites.

Propargyl-PEG2-NHS ester is a PEG linker with a propargyl group that can participate in copper catalyzed azide-alkyne Click Chemistry and NHS ester that can be used to label amine-containing entitites.

Overview

• Isolate all sizes of circulating and exosomal RNA, including microRNA
• Versatile plasma/serum input ranges
• No phenol extractions
• No carrier RNA
• Bind and elute all RNA irrespective of size or GC content, without bias
• Concentrate circulating RNA and exosomal RNA into a flexible elution volume
• High quality, purified RNA is suitable for a variety of downstream applications, including Small RNA Sequencing. Find out more information on Norgen’s NGS services
• Compatible with Streck Cell-Free RNA BCT® Tubes
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

These kits provide a fast, reliable and convenient method to purify and concentrate high quality, high purity and inhibitor-free cell-free circulating and exosomal RNA using a convenient spin column method. These kits can purify RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate. Plasma samples prepared from blood collected on heparin should not be used, as heparin can significantly interfere with many downstream applications such as RT-PCR. The purified plasma/serum RNA is fully compatible with all downstream applications including PCR, qPCR, methylation-sensitive reverse transcription qPCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, expression array assays, and NGS.

Background

Plasma/Serum cell-free circulating RNA or exosomal RNA has the potential to provide biomarkers for certain cancers and disease states. Exosomes are 40 – 150 nm membrane vesicles, which are secreted by most cell types. Exosomes can be found in saliva, blood, urine, amniotic fluid and malignant ascitic fluids, among other biological fluids. Evidence has been accumulating recently that these vesicles act as cellular messengers, conveying information to distant cells and tissues within the body. These exosomes may play a functional role in mediating adaptive immune responses to infectious agents and tumours, tissue repair, neural communication and transfer of pathogenic proteins. For this reason exosomal RNAs may serve as biomarkers for various diseases including cancer. As the RNA molecules encapsulated within exosomes are protected from degradation by RNAses they can be efficiently recovered from biological fluids, such as plasma or serum.

Plasma/Serum RNA Purification Mini Kit

This kit can purify RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate, from volumes ranging from 50 µL to 200 µL. The purified plasma/serum RNA is eluted in a flexible final volume of 10 µL to 25 µL.

Plasma/Serum RNA Purification Midi Kit

This utilizes a two column method, and can purify RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate, from volumes ranging from 250 µL to 1.5 mL. The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 50 µL to 100 µL.

Plasma/Serum RNA Purification Maxi Kit

This kit can purify RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate, from volumes ranging from 2 mL to 5 mL. The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 50 µL to 100 µL.

Isolate RNA after Purifying EVs and Exosomes

For Ultracentrifugation, Exoquick, and Filtration

Cat. #	Name	Elution Volume	Plasma/Serum	Urine	Cell-Culture Media
55000	Plasma/Serum RNA Purification Mini Kit	10 – 25 µL	50 µL – 1 mL	250 µL – 1 mL	5 – 10 mL
35300	Total RNA Purification Micro Kit	20 – 50 µL	1 – 4 mL	2 – 10 mL	10 – 20 mL
17200	Total RNA Purification Kit	50 – 100 µL	4 – 10 mL	11 – 30 mL	20 – 35 mL

Details

Supporting Data

Figure 1 / 13

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Kit Specifications
Minimum Plasma/Serum Input	2 mL
Maximum Plasma/Serum Input	5 mL
Size of DNA Purified	All sizes, including miRNA and small RNA (<200 nt)
Elution Volume	50-100 μL
Time to Complete 10 Purifications	35-40 minutes
Average Yields*	Variable depending on specimen

*Please check page 7 of the Product Insert for Average Plasma/Serum Yields and Common RNA Quantification Methods.

This kit is suitable for the isolation of RNA from fresh or frozen serum or plasma prepared from blood collected on either EDTA or Citrate. Plasma samples prepared from blood collected on heparin should not be used as heparin can significantly interfere with many downstream applications such as RT-PCR.
 

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.

Kit Components	Cat. 55000 (50 preps)	Cat. 56100 (20 preps)	Cat. 56200 (10 preps)
Lysis Buffer A	2 x 20 mL	100 mL	1 x 130 mL 1 x 30 mL
Wash Solution A	18 mL	1 x 38 mL 1 x 18 mL	38 mL
Elution Solution A	6 mL	6 mL	6 mL
Elution Buffer F	–	15 mL	15 mL
Micro Spin Columns	50	–	–
Mini Spin Columns	–	20	10
Midi Spin Columns	–	20	–
Maxi Spin Columns	–	–	10
Collection Tubes	50	20	10
Elution Tubes (1.7 mL)	50	20	10
Product Insert	1	1	1

Endonucleases DNA-specific, dsDNase

OverView

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.