TE buffer

Last updated

TE buffer is a commonly used buffer solution in molecular biology, especially in procedures involving DNA, cDNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that chelates cations like Mg2+. The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.

Contents

Recipe

A typical recipe for making 1X TE buffer is:

TE buffer is also known as T10E1 buffer, which can be read as "T ten E one buffer". To make a 100 ml solution of T10E1 buffer, 1 ml of 1 M Tris base (pH 10–11) and 0.2 ml EDTA (0.5 M) are mixed and made up with double distilled water up to 100ml. Add microliter amounts of high molarity HCl to lower the pH to 8.

Based on nuclease studies from the 1980s, the pH is usually adjusted to 7.5 for RNA and 8.0 for DNA.[ citation needed ] The respective DNA and RNA nucleases are supposed to be less active at these pH values, but pH 8.0 can safely be used for storage of both DNA and RNA [ citation needed ].

EDTA further inactivates DNase, by binding to metal cations required by this enzyme. [1]

Genomic and plasmid DNA can be stored in TE Buffer at 4 °C (39.2 °F) for short-term use, or -20 °C (-4 °F) to -80 °C (-112 °F) for long-term storage. Repeated freeze-thaw cycles should be avoided. [2]

Low TE or TE Low EDTA

The operation of the TE buffer is based on chelating metal cations such as Mg2+. The problem is that the PCR polymerase also requires Mg2+ to function, so if the amount of EDTA is too high it can affect the PCR. There is a version of TE buffer with 10 times less amount of EDTA that is very frequently used for the forensic STR kits. Due to the use of kits with multiplex amplification, it is necessary to have a lower amount of EDTA in the sample so as not to interfere with the Mg2+ present in the reaction. If regular TE buffer is used to dilute the sample, an imbalance will be observed in the DNA profile, while Low TE will have a better balance. The Low TE buffer or TE Low EDTA buffer is composed of 10 mM Tris-HCl (pH 8.0) + 0.1 mM EDTA.

See also

Related Research Articles

<span class="mw-page-title-main">Agarose gel electrophoresis</span> Method for separation and analysis of biomolecules using agarose gel

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size, and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.

<span class="mw-page-title-main">Ethylenediaminetetraacetic acid</span> Chemical compound

Ethylenediaminetetraacetic acid (EDTA), also called edetic acid after its own abbreviation, is an aminopolycarboxylic acid with the formula [CH2N(CH2CO2H)2]2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes even at neutral pH. It is thus used to dissolve Fe- and Ca-containing scale as well as to deliver iron ions under conditions where its oxides are insoluble. EDTA is available as several salts, notably disodium EDTA, sodium calcium edetate, and tetrasodium EDTA, but these all function similarly.

Viral load, also known as viral burden, is a numerical expression of the quantity of virus in a given volume of fluid, including biological and environmental specimens. It is not to be confused with viral titre or viral titer, which depends on the assay. When an assay for measuring the infective virus particle is done, viral titre often refers to the concentration of infectious viral particles, which is different from the total viral particles. Viral load is measured using body fluids Sputum and blood plasma. As an example of environmental specimens, the viral load of norovirus can be determined from run-off water on garden produce. Norovirus has not only prolonged viral shedding and has the ability to survive in the environment but a minuscule infectious dose is required to produce infection in humans: less than 100 viral particles.

<span class="mw-page-title-main">Reverse transcription polymerase chain reaction</span> Laboratory technique to multiply an RNA sample for study

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

<span class="mw-page-title-main">His-tag</span>

A polyhistidine-tag, best known by the trademarked name His-tag, is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as a hexa histidine-tag, 6xHis-tag, or His6 tag. The tag was invented by Roche, although the use of histidines and its vectors are distributed by Qiagen. Various purification kits for histidine-tagged proteins are commercially available from multiple companies.

Deoxyribonuclease refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells includes breaking down extracellular DNA (ecDNA) excreted by apoptosis, necrosis, and neutrophil extracellular traps (NET) of cells to help reduce inflammatory responses that otherwise are elicited. A wide variety of deoxyribonucleases are known and fall into one of two families, which differ in their substrate specificities, chemical mechanisms, and biological functions. Laboratory applications of DNase include purifying proteins when extracted from prokaryotic organisms. Additionally, DNase has been applied as a treatment for diseases that are caused by ecDNA in the blood plasma. Assays of DNase are emerging in the research field as well.

A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells. Most lysis buffers contain buffering salts and ionic salts to regulate the pH and osmolarity of the lysate. Sometimes detergents are added to break up membrane structures. For lysis buffers targeted at protein extraction, protease inhibitors are often included, and in difficult cases may be almost required. Lysis buffers can be used on both animal and plant tissue cells.

The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.

TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA.

Phosphate-buffered saline (PBS) is a buffer solution commonly used in biological research. It is a water-based salt solution containing disodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and potassium dihydrogen phosphate. The buffer helps to maintain a constant pH. The osmolarity and ion concentrations of the solutions match those of the human body (isotonic).

<span class="mw-page-title-main">Bis-tris propane</span> Chemical compound

Bis-tris propane, or 1,3-bis(tris methylamino)propane, also known as BTP, is a chemical substance that is used in buffer solutions. It is a white to off-white crystalline powder that is soluble in water. It has a wide buffering range, from 6 to 9.5 due to its two pKa values which are close in value. This buffer is primarily used in biochemistry and molecular biology.

TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.

Mung bean nuclease is a nuclease derived from sprouts of the mung bean that removes nucleotides in a step-wise manner from single-stranded DNA molecules (ssDNA) and is used in biotechnological applications to remove such ssDNA from a mixture also containing double-stranded DNA (dsDNA). This enzyme is useful for transcript mapping, removal of single-stranded regions in DNA hybrids or single-stranded overhangs produced by restriction enzymes, etc. It has an activity similar to Nuclease S1, but it has higher specificity for single-stranded molecules.

Alkaline lysis or alkaline extraction is a method used in molecular biology to isolate plasmid DNA from bacteria.

<span class="mw-page-title-main">Molecular-weight size marker</span> Set of standards

A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.

Chelex 100 is a chelating material from Bio-Rad used to purify other compounds via ion exchange. It is noteworthy for its ability to bind transition metal ions.

<span class="mw-page-title-main">Proteinase K</span>

In molecular biology Proteinase K is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Parengyodontium album. Proteinase K is able to digest hair (keratin), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8 (subtilisin). The molecular weight of Proteinase K is 28,900 daltons.

TSE or Tris/Saline/EDTA, is a buffer solution containing a mixture of Tris base, Sodium chloride and EDTA.

Ferric reducing ability of plasma is an antioxidant capacity assay that uses Trolox as a standard. The FRAP assay was first performed by Iris Benzie and J. J. Strain of the Human Nutrition Research Group at the University of Ulster, Coleraine. The method is based on the formation of O-Phenanthroline-Fe(2+) complex and its disruption in the presence of chelating agents. This assay is often used to measure the antioxidant capacity of foods, beverages and nutritional supplements containing polyphenols.

Serratia marcescens nuclease is an enzyme. This enzyme catalyses the following chemical reaction

References

ph7.4 TE buffer=100mM/L Tris(pH7.4)+10mM/L EDTA(pH8.0) from Molecular Cloning: A Laboratory Manual

  1. ↑ Yagi N, Satonaka K, Horio M, Shimogaki H, Tokuda Y, Maeda S (1996). "The role of DNase and EDTA on DNA degradation in formaldehyde fixed tissues". Biotechnic & Histochemistry. 71 (3): 123–129. doi:10.3109/10520299609117148. PMID   8724437.
  2. ↑ Ross; Haites, Kelly (1990). "Repeated freezing and thawing of peripheral blood and DNA in suspension: effects on DNA yield and integrity". Journal of Medical Genetics. 27 (9): 569–570. doi:10.1136/jmg.27.9.569. PMC   1017219 . PMID   2231649.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.