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Manual Nucleic Acid Isolation & Purification
Purification or isolation of nucleic acids is the first step in most molecular biology studies and all recombinant DNA techniques.
High Pure Nucleic Acid Isolation
Start your experiments right - generate high-quality nucleic acids with Roche isolation and purification kits. Roche offers a wide array of products for DNA or RNA preparations, from amultitude of sample material (see Selection Table). Use the Application Guide to choose the right kit for your downstream applications.
- Process more samples in less time.
- Minimize nucleic acid loss and degradation.
- Increase laboratory efficiency and safety.
- Avoid organic solvents and toxic reagents.
- Isolate nucleic acids from different sample materials.
- Purify nucleic acids of highest integrity, suitable for demanding down-stream applications.
Isolation and Purification of DNA - Find Products
Isolation and Purification of RNA - Find Products
Isolation and Purification of Total Nucleic Acids - Find Products
Isolation and Purification before and after PCR - Find Products
Columns for Purification
DNA Purification Products
- PCR/Long PCR
- Restriction Enzyme Digestion
- Southern Blotting
- Labeling, Modifying Reactions
RNA Purification Products
- Differential Display RT-PCR
- cDNA Synthesis/Primer Extension
- Northern Blotting
- RNase Protection Assays
- In vitro Translation
Total Nucleic Acid Purification Products
- PCR/Long PCR
- Differential Display RT-PCR
- cDNA Synthesis/Primer Extension
- Southern Blotting
- Northern Blotting
- RNase Protection Assays
The extraction of nucleic acids from biological material requires cell lysis, inactivation of cellular nucleases, and separation of the desired nucleic acid from cellular debris. Conventional methods usually employ a lysis procedure which is rigorous enough to fragment the complex starting material (e.g., blood or tissue) and inactivate nucleases, yet gentle enough to preserve the target nucleic acid.
Traditional methods for purifying nucleic acids from cell extracts are often combinations of extraction, precipitation, chromatography, centrifugation, electrophoresis, and affinity separation. Unfortunately, most of these methods require extensive handling of toxic chemicals (e.g., phenol or ethidium bromide), need expensive equipment (e.g., ultracentrifuges), and are time consuming. To minimize these problems, Roche has developed an extensive line of versatile nucleic acid isolation and purification products.
To learn more about the characteristics of these products, follow the links below:
High Pure Kit and Silica Adsorption Kit
Combine centrifugation, chromatography on glass fiber fleece or silica beads, and chaotropic salt extraction. These rapid purification kits eliminate traditional solvent extraction, precipitation, and electrophoresis steps.
Ion Exchange Chromatography
Employ different salt conditions for binding and release of nucleic acids. Solutions are simply poured or pipeted into the matrix-filled columns, which are run by gravity flow. The DNA obtained by this method has purity comparable to that obtained when purified twice by cesium chloride centrifugation.
Solution-Based Isolation Kits and Reagents
Use proprietary cell lysis and extraction methods that are quicker and safer than standard methods. These products prepare nucleic acids with minimal handling.
Affinity Purification Kits
Exploit the hybridization properties of nucleic acids. These products eliminate time-consuming centrifugation and electrophoresis steps.
Affinity purification is a versatile and highly specific technique for the purification of all classes of biomolecules utilizing differences in biological activities of chemical structures. The high selectivity of this technique results in good purification and high recovery. Often a concentrating effect is reached which enables large volumes to be conveniently processed.
The mRNA kits rely on base pairing between poly(A+) residues at the 3'-end of mRNAs and the oligo(dT) residues of a biotin-labeled oligo(dT) probe. The biotinylated dT-A hybrid is bound to streptavidin-coated surfaces of either tubes or magnetic particles. Some of the kits prepare mRNA directly whereas the other ones start with the purification of total nucleic acid and subsequent isolation of the mRNA.
Products that employ affinity purification or work in conjunction with these products are > Product list
Gel Filtration Columns
Rely on ready-to-use spin columns. These columns take just minutes to separate nucleic acids from salts, unincorporated nucleotides, or excess primers.
The products described in this section depend on gel filtration chromatography, which separates molecules based on their relative size. This type of chromatography was originally described by Porath and Flodin [Porath , J. and Flodin, P. (1959) Nature 183, 1657-1659] for the desalting of proteins.
If a molecule can enter and exit the pores of the gel matrix, its rate of movement is determined by the flow of the chromatographic buffer and the diffusion properties of the molecule. The matrix thus functions like a molecular sieve. Smaller molecules enter and leave many pores of the matrix, thus traversing the length of the column relatively slowly. Larger molecules do not enter the gel pores and therefore elute rapidly from the column.
Roche has adapted gel filtration technology to a spin column format referred to as the Quick Spin method for use in a wide range of applications. The mini Quick Spin DNA Columns, mini Quick Spin RNA Columns, and mini Quick Spin Oligo Columns are specially designed for the removal of unincorporated nucleotides, fluorescent dye-labeled dideoxy terminators, or other small contaminating molecules from labeled RNA, DNA, or oligonucleotides, respectively. The columns are packed with a prehydrated Sephadex matrix, ready-for-use, and operate by simple spin column centrifugation using a conventional tabletop microcentrifuge.
The mini Quick Spin Columns and Quick Spin Columns contain gel filtration matrices (either G-25 or G-50 Sephadex) which allow large molecules (e.g., DNA or RNA) to pass through quickly while retaining small molecules (e.g., nucleotides). The Quick Spin format improves the molecular sieving concept by using centrifugation to separate DNA or RNA rapidly and cleanly from small contaminants.
mini Quick Spin Columns and Quick Spin Columns are:
Prespin column, apply an undiluted sample to the column bed, and centrifuge the column for a few minutes to recover the purified DNA or RNA.
Both types of columns can separate unincorporated nucleotides from labeled DNA or RNA in a few minutes.
The columns are ready-to-use and are even supplied with collection tubes.
Columns are quality tested to ensure the absence of nucleases, maximum recovery of nucleic acid, and maximum retention of small contaminating molecules.
For further information on the product most relevant to your research, please follow the respective link.
Success of most procedures in molecular biology depends on the purity, integrity, and exact quantification of nucleic acids. In this section, we present general guidelines for handling and quantifying nucleic acids. These tips are not intended to cover these topics in detail, but to identify important areas which should be considered when performing experimental procedures.
- Precautions for Handling of DNA
- Precautions for Handling of RNA
- Spectrophotometric Quantitation of DNA and RNA
Handling fresh and stored material before extraction of DNA
For the isolation of genomic DNA from cells or tissues, use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at -70°C. This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases. Follow this procedure for both genomic DNA and plasmid DNA.
Storage of DNA
Store genomic DNA at +2 to +8°C. Storing genomic DNA at -15 to -25°C can cause shearing of DNA, particularly if the DNA is exposed to repeated freeze-thaw cycles. Plasmid DNA and other small circular DNAs can be stored at +2 to +8°C or at -15 to -25°C.
Genomic and plasmid DNA can also be stored in small aliquots. Repeated use of a single sample may lead to shearing.
Drying, dissolving and pipetting DNA
Avoid overdrying genomic DNA after ethanol precipitation. It is better to let it air dry than to use a vacuum, although vacuum drying can be used with caution. Plasmid DNA and other small circular DNAs can be vacuum dried.
To help dissolve the DNA, carefully invert the tubes several times after adding buffer and or tap the tube gently on the side. Alternatively let the DNA stand in buffer overnight at +2 to +8°C. Minimize vortexing of genomic DNA since this can cause shearing.
Avoid vigorous pipetting. Pipetting genomic DNA through small tip openings causes shearing or nicking. One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA. Regular pipette tips pose no problem for plasmid DNA and other small circular DNAs.
Nature of RNA
In contrast to DNA, RNA is a single-stranded polynucleotide that is very susceptible to degradation by base- or enzyme-catalyzed hydrolysis. Working with RNA is more demanding due to both the chemical instability of RNA and because of the ubiquitous presence of RNases. Further, unlike DNases which require metal ions for activity, RNases have no requirement for metal ion co-factors and can maintain activity even after prolonged boiling or autoclaving.
RNases are found everywhere, including on laboratory workers´ hands and in airborne microorganisms. Special precautions must be taken when working with RNA. All reagents and equipment must be specially treated to inactivate RNases prior to use. Below are some tips to help ensure that your laboratory environment is as clean as possible.
Tips for Maintaining an RNase-free Environment
Gloves and contact
When working with RNA, wear gloves at all times. After putting on gloves, avoid touching contaminated surfaces and equipment with the gloved hands. Even if all the reagents have been decontaminated, RNases can be reintroduced by contact with ungloved hands or with unfiltered air.
Equipment and disposable items
Use sterile, disposable plasticware whenever possible. These require no treatment and are considered to be RNase-free. Electrophoresis tanks for RNA analysis can be cleaned by wiping them with a solution of SDS (1%), rinsing with water, then rinsing with absolute ethanol, and finally soaking them in 3% H2O2 for 10 minutes. Rinse tanks with DEPC (diethyl pyrocarbonate)-treated and autoclaved water before use (see below).
Glassware and plasticware
Treat glassware and plasticware with RNase-inactivating agents. Glassware should be baked at +180°C for at least 4 hours. Note, however, that autoclaving alone is not sufficient to eliminate RNases from your experiments. Soak plasticware (2 hours, +37°C) in 0.1 M NaOH/1 mM EDTA (or absolute ethanol with 1% SDS), rinsed with DEPC or DMPC (dimethyl pyrocarbonate) treated water and heated to +100°C for 15 minutes in an autoclave.
To treat water with DEPC (or DMPC, dimethyl pyrocarbonate, a less toxic alternative to DEPC that can be used in the same manner as DEPC), first incubate it with DEPC (2 hours, +37°C) and then autoclave it to hydrolyze any unreacted DEPC.
Workspace and working surfaces
Designate a special area for RNA work only. Treat surfaces of benches and glassware with commercially available RNase inactivating agents. Also, wipe benches with 100% ethanol each time prior to use, in order to rid the area of microorganisms.
Whenever possible, purchase reagents that are free of RNases. Be sure to separate reagents used for RNA work from "general use reagents" in the laboratory. All solutions, except Tris buffers, should be treated with 0.1% DEPC (or DMPC) overnight at room temperature and then autoclaved. Autoclaving hydrolyzes and destroys unreacted DEPC and DMPC. Alternatively, solutions can be made with DEPC-treated and autoclaved water in RNase-free glassware. Tris reacts chemically with DEPC and DMPC, and therefore, solutions of Tris cannot be made RNase-free using DEPC or DMPC. Dedicate one bottle of Tris for RNA work only. Use baked spatulas and glassware and DEPC/DMPC-treated water for making the buffers.
Note: Autoclaving without DEPC/DMPC treatment is insufficient for inactivating RNases.
Precautions for handling RNA
Handling of fresh and stored material before extraction of RNA
Extract RNA as quickly as possible after obtaining samples. For the best results, use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at -70°C. RNA in inadequately maintained samples can be degraded by intracellular nucleases, specifically in tissues that are rich in nucleases (such as spleen and pancreas).
RNase inhibitors can be used to protect RNA from degradation during both isolation and purification and also in downstream applications such as reverse transcription into cDNA by RT-PCR, in vitro RNA transcription/translation reactions and RNA-dependent in vitro functional assays.
Protector RNase Inhibitor
Protector RNase Inhibitor is one of the best characterized RNase inhibitors, effective on a wide spectrum of RNases (RNase A, RNase B, RNase T2) and offering the best protection of precious RNA samples. Protector RNase Inhibitor is fully active over a broad temperature range of +25 to +55°C. Even at +60°C some RNase inhibition is still measured. This is advantageous when performing reverse transcription reactions at elevated temperatures to overcome secondary structure in RNA, or when working with thermostable reverse transcriptases like Transcriptor Reverse Transcriptase. To keep the inhibitor active, avoid temperatures above +60°C, solutions containing strong denaturing agents (such as SDS or urea), and maintain reducing conditions (1 mM DTT). To protect difficult RNA samples, the amount of Protector RNase Inhibitor can be increased up to 16 times the standard concentration without interfering with the performance of enzymes used in the assay.
Other inhibitors include Macaloid and Vanadyl-ribonucleoside complexes.
Storage of RNA
Store RNA at -70 to -80°C, as aliquots in ethanol or isopropanol. Most RNA is relatively stable at this temperature. Centrifuge the RNA and resuspend in an appropriate RNase-free buffer before use.
Drying, dissolving, and pipeting RNA
RNA can be dried briefly at +37°C or in a vacuum oven. When working with RNA, place all samples on ice. For the reasons mentioned above, RNA is very susceptible to degradation when left at room temperature.
Dissolve RNA by adding RNase-free buffer or water, then standing the tube on ice for 15 minutes. Gently tap the tube or use vortexing with caution.
Although DNA is relatively stable at elevated temperatures (+100°C), most RNA is not (except for short RNA probes, which are stable for 10 minutes at +100°C).
Therefore, avoid high temperatures (above +65°C) since these affect the integrity of the RNA. Instead, to melt out secondary structures, heat RNA to +65°C for 15 minutes in the presence of denaturing buffers.
|Nucleic Acid||Amount / 1 A260 Unit||Molarity (Nucleotides) / 1 A260 Unit|
|Double-stranded DNA||50 µg/ml||0.15 mM|
|Single-stranded DNA||33 µg/ml||0.10 mM|
|Single-stranded RNA||40 µg/ml||0.11 mM|
|Oligonucleotide||20 - 30 µg/ml||0.06 - 0.09 mM|
Determining the purity of nucleic acid preparations
For pure DNA: A260 / A280 = 1.8
For pure RNA: A260 / A280 = 2.0