6-Formyl-2-pyridine-PEG4-DBCO is a bifunctional PEG linker featuring an aldehyde and a DBCO function. DBCO is a strained cyclooctyne which reacts with azide groups on target molecules while the aldehyde group is a versatile electrophile that is reactive towards amines, hydroxylamines, and hydrazines.

6-Formyl-2-pyridine-PEG4-DBCO is a bifunctional PEG linker featuring an aldehyde and a DBCO function. DBCO is a strained cyclooctyne which reacts with azide groups on target molecules while the aldehyde group is a versatile electrophile that is reactive towards amines, hydroxylamines, and hydrazines.

Introduction

The Kit is designed for purification of total RNA, including miRNA and other small RNA molecules (18nt), from cultured cells and various animal and human tissues, including difficult-to-lyse tissues samples.

Details

Specifications

Features	Specifications
Main Functions	Isolation total RNA and miRNA from cell and tissue without MagZol reagent
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	Cell, Animal tissue, Plant tissue
Sample amount	Cells: ≤ 5 x 10^6, Animal tissue:<10mg
Elution volume	≥30μl
Time per run	≤40 minutes
Liquid carrying volume per column	800µl
Binding yield of column	100µg

Principle

Biological samples are first lysed and homogenized in a highly denaturing guanidine isothiocyanate-containing buffer, which immediately inactivates DNases and RNases to ensure isolation of intact DNA and RNA. The lysate is then passed through a Mini spin column. This column, in combination with the high-salt buffer, allows selective and efficient binding of genomic DNA. Flow-through from the column is digested by Proteinase K in the presence of ethanol. This optimized digestion, together with the subsequent addition of further ethanol, allows appropriate binding of total RNA, including miRNA, to the column. Contaminants are efficiently washed away and high-quality RNA is eluted.

Advantages

• High quality – one step RNA extraction reagent combined with silica gel column can obtain the highest concentration
• Fast – several samples can be extracted in 40 minutes
• High applicability – samples including animals, plants, bacteria, cells, etc.
• High concentration – efficiently remove macromolecular RNA, enrich small RNA and improve sensitivity
• Safe – no phenol chloroform extraction and no use of Trizol reagent

Kit Contents

Contents	R431102	R431103
Purification Times	50 Preps	250 Preps
HiPure RNA Mini Columns	100	2 x 250
2ml Collection Tubes	100	2 x 250
Proteinase K	48 mg	240 mg
Protease Dissolve Buffer	5 ml	15 ml
Buffer RLC	40 ml	200 ml
Buffer RWC	20 ml	80 ml
Buffer RW2*	20 ml	2 x 50 ml
RNase Free Water	10 ml	60 ml

Storage and Stability

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

The Kit is designed for purification of total RNA, including miRNA and other small RNA molecules (18nt), from cultured cells and various animal and human tissues, including difficult-to-lyse tissues samples.

Overview

• Sequentially purify total RNA and total proteins from a single sample
• Kit includes a gDNA elimination column  
• No sample splitting required
• No phenol step required for efficient isolation
• Ideal for small or difficult to obtain samples
• Purify RNA and proteins from cultured animal cells, tissues, blood, bacteria, yeast, fungi or plants
• Rapid and efficient spin column procedure
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

This kit provides a rapid, single column method for the isolation and purification of total RNA (including miRNA) and proteins sequentially from a single sample of cultured animal cells, tissues, blood, bacteria, yeast, fungi or plants. The total RNA and proteins are both column purified in under 25 minutes using a single column.

Purified RNA is of a high quality and yield, and is suitable for NGS, RT-qPCR and microarrays.  

This kit eliminates DNA efficiently using a gDNA removal column.

Proteins are eluted in buffer and are ready for downstream applications such as Western Blots, Mass Spec and ELISA.  The proteins will not require precipitation, resuspending of pellets, or any further cleaning.

This kit is ideal for researchers who are interested in studying the transcriptome and proteome of a single sample, such as for studies of microRNA profiling, gene expression including gene silencing experiments or mRNA knockdowns, studies involving biomarker discovery, and for characterization of cultured cell lines. Norgen’s RNA/Protein Purification Plus Kit is especially useful for researchers who are isolating macromolecules from precious, difficult to obtain or small samples such as biopsy materials or single foci from cell cultures, as it eliminates the need to fractionate the sample. Furthermore, analysis will be more reliable since the RNA and proteins are derived from the same sample, thereby eliminating inconsistent results. The purified macromolecules are of the highest purity and can be used in a number of different downstream applications.

Protocol

Details

Supporting Data

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Kit Specifications
Maximum Column Binding Capacity	50 μg for RNA
Maximum Column Binding Capacity	200 μg for Protein
Maximum Column Loading Volume	650 μL
Size of RNA Purified	All sizes, including small RNA (< 200 nt)
Time to Complete 10 Purifications	30 minutes
Average Yields*: HEK 293 Cells (1 x 106 cells) HEK 293 Cells (1 x 106 cells) Liver (15 mg) Liver (15 mg)	10-15 μg RNA 70-100 μg protein 30-35 μg RNA 100-150 μg protein

* Average yields will vary depending upon a number of factors including species, growth conditions used and developmental stage.

Storage Conditions and Product Stability
The Protein Loading Dye should be stored at -20°C after the addition of DL-Dithiothreitol (DTT). All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.

Component	Cat. 48200 (50 preps)
Buffer SKP	40 mL
Wash Solution A	38 mL
Elution Solution A	6 mL
Wash Solution C	30 mL
Binding Buffer A	8 mL
Elution Buffer C	4 mL
Protein Neutralizer	4 mL
Protein Loading Dye	2 mL
gDNA Removal Columns	50
RNA/Protein Purification Columns	50
Collection Tubes	150
Elution Tubes (1.7 mL)	100
Product Insert	1