Nucleic Acid Purification Protocols

A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue Protocol for a single-step method for the simultaneous preparation of DNA, RNA, and protein from cells and tissues. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O.

Concentrating and Desalting Nucleic Acids with Microconcentrators Protocol Ultrafiltration is an alternative to ethanol precipitation for the concentration and desalting of nucleic acid solutions.  It requires no phase change and is particularly useful for dealing with very low concentrations of nucleic acids.  This protocol describes the use of the Microcon cartridge, a centrifugal ultrafiltration device, to concentrate and desalt nucleic acid solutions.

Mapping RNA with Nuclease S1 Protocol Preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe.  At the end of the reaction nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by autoradiography or Southern hybridization.  Method used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates.

Oligonucleotide Purification Protocol Protocol for oligonucleotide purification.

Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters Protocol Protocol for purification of in vitro synthesized mRNA with Microcon or Centricon Centrifugal Filters.

Purification of Labeled Probes using the High Pure PCR Product Purification Kit Protocol Protocol for purification of labeled probes using the High Pure PCR Product Purification Kit.

Purification of Nucleic Acids by Extraction with Phenol:Chloroform Protocol Protocol describes the standard method for nucleic acid purification by extraction first with phenol:chloroform (optionally containing hydroxyquiniline at 0.1%) and then with chloroform to remove any remaining phenol.  The procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one.

Purification of Radiolabeled Oligonucleotides by Precipitation with Cetylpyridinium Bromide Protocol Radiolabeled nucleic acids, including oligonucleotides, can be separated from unincorporated radiolabel by quantitative, differential precipitation with the cationic detergent cetylpyridinium bromide (CPB).  The nucleic acids are first precipitated from aqueous solution with CPB.

Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration Protocol In this protocol, ultrafiltration through Centricon or Microcon concentrators is used to remove unused primers, primer-dimers, and NTPs from preparations of amplified DNA.

Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes Protocol Protocol describes the standard method to recover nucleic acids from aqueous solutions by precipitation of DNA with ethanol.  Subnanogram amounts of DNA (and RNA) can be quantitatively precipitated with ethanol, collected by centrifugation, and redissolved within minutes.