Introduction

Nucleic acid extraction widely encompasses the processes of cell collection, lysis, nucleic acid isolation, purification, and recovery. Samples entering clinical or research laboratories for nucleic acid extraction range from blood and saliva, to fresh, frozen, or decades-old archived tissue biopsies, to aquatic samples and soils of environmental or agricultural interest.

Evaluation of extraction quality is ultimately functional. A successful extraction is one that yields nucleic acid material of sufficient quantity, quality, purity, and integrity that it can be used as substrate material for the intended laboratory protocol.

DNA Isolation

Filter column purification can be used to purify DNA from mammalian cell cultures, bacteria, and yeast, as well as plant and animal tissue. By adjusting the pH and salt of the solution, DNA of interest can be separated from cellular debris or other unwanted contaminants by binding the DNA to a silica membrane situated at the bottom of a filter column. This is a popular method for genomic and plasmid DNA purification. Vacuum and centrifugation protocols are available. After the DNA is bound to the membrane, it is subjected to a low and a high stringency wash to remove contaminants such as RNA, proteins and lipids. The purified DNA is eluted off the membrane with an elution buffer into a collection tube. The volume of the elution buffer used can be varied depending on the final concentration of plasmid or DNA desired. Recovered DNA is suitable for use with PCR and Southern blot analysis.

RNA Isolation

When working with cells or tissues as the starting material, the first step of the nucleic acid purification process is cell lysis or membrane permeabilization. This process breaks open the cell membranes and disrupts the cellular structure to create a cell lysate. This allows the nucleic acid of interest to be accessed and separated away from unwanted cellular components. Cell lysis can be achieved either by mechanical techniques such as grinding, douncing, and sonicating, or it can be performed chemically with a lysis buffer. Lysis buffers are designed to rupture cells by osmosis and their compositions can be tailored for specific applications. Lysis buffers typically contain a detergent such as NP-40 or Triton to disrupt membrane lipids. Chelating agents such as EDTA and EGTA may also be present in the lysis buffer to inhibit nuclease activity. Proteases such as proteinase K can also be used to facilitate cell lysis. The resulting cell lysate can then be subjected to a physical separation such as centrifugation, a chemical separation such as phenol/chloroform extraction, or a solid phase-based separation such as a column to isolate the nucleic acid of interest.

Services

• DNA Extraction & Purification
• DNA Nucleases & Glycosylases
• DNA Polymerases
• RNA Extraction & Purification

Specifiation

• DNA Isolation

• RNA Extraction