RNase Contamination Assay Kit

  • This product was discontinued on 01/01/2016
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Description

Evaluation of RNase contamination is necessary for reagents to be used in experiments with RNA. The RNase Contamination Assay Kit detects general RNase activities including non-enzyme based RNA degradation due to heavy metal contamination in samples and high pH. The assay probe is a fluorescein labeled RNA transcript (300-mer). After incubation with a reagent sample the integrity of the RNA probe is analyzed on denaturing PAGE followed by SYBR Gold staining or preferably by scanning with a FAM/Fluorescein capable imaging system like the Typhoon scanner. Most samples (e.g. enzymes, column fractions from enzyme purifications, unlabeled RNA/DNA samples, buffers, or diluents) can be tested directly. The assay is semi-quantitative and is more sensitive if detergent is present.

In addition to RNase assay, the labeled RNA probe can also be used in studies involving Poly(A) and Poly(U) tailing, RNA ligation, RNA capping/decapping assays and sequence and structure specific RNase assays.

Materials not included:
Nuclease-free water, incubator, PAGE/Urea gel, TBE running buffer, power supply, SYBR Gold, Typhoon scanner and UV light box.

Fluorescein Labeled RNA Probe:
The probe is an in vitro RNA transcript (300-mer), internally labeled with Fluorescein-12-UTP, supplied at 40 ng/µl.

RNA sequence:
1 GGGAAGAGCA UUGUCGAGGG UGAAGUACGG UUCUUGCAGG UUGAACAUCA GCGCGCUCUU
61 ACCUUUCGCU UUCAGUUCUU UAUCCAGCGC CGGGAUCUCU UCCCAGGUUU UUGGCGGGUU
121 CGGCAGCAGA UCUUUGUUAU AAAUCAGCGA UAACGCUUCA ACAGCGAUCG GGUAAGCAAU
181 CAGCUUGCCG UUGUAACGUA CGGCAUCCCA GGUAAACGGA UACAGCUUGU CCUGGAACGC
241 UUUGUCCGGG GUGAUUUCAG CCAACAGGCC AGAUUGAGCG UACUCGACGA AGACUCCCUA


1X NEBuffer 4:
50 mM Potassium acetate
20 mM Tris-acetate
10 mM Magnesium acetate
1 mM Dithiothreitol
(pH 7.9 at 25°C)

2X RNA Loading Dye:
95% Formamide
0.02% SDS
0.02% Bromophenol blue
0.01% Xylene cyanol
1 mM EDTA

Proteinase K:
4 mg/ml

Figure 1A:
Figure 1A

Proteinase K treatment releases bound RNA probe. After incubation for 16 hours at 37°C, control and test reactions were analyzed on 6% PAGE-Urea gel. The gel was imaged on a Typhoon scanner. PK treatment releases bound RNA probe (reactions 1 and 2). The +PK panel shows reactions 2 and 3 with intact RNA probe, indicating that the samples are clean. Reaction 1 shows heavy degradation with free label at the bottom, indicating that the sample is contaminated with RNase activity.
Figure 1B:
Figure 1B

The same gel in Figure 1A stained with SYBR Gold. Note the free label in reaction 1 is less visible.

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
Fluorescein-RNA Probe40 μg/ml
RNA Loading Dye, (2X)2X
NEBuffer 4-2010X
Proteinase K, Molecular Biology Grade-204 mg/ml

Advantages and Features

Features

Convenient gel-based assay
RNase contamination is clearly visualized
Can be used with any reagent not interfering with fluorescein imaging
Can be loaded directly on gel

Applications

Evaluation of RNase contamination

Properties and Usage

Storage Temperature

-20°C

Protocols

  1. RNase Contamination Assay Kit (E3320)

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

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Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.