TE buffer, commonly known as Tris-EDTA buffer, is a widely used aqueous solution in molecular biology for the solubilization, storage, and protection of nucleic acids such as DNA and RNA.[1] It typically consists of 10 mM Tris-HCl adjusted to pH 8.0 and 1 mM EDTA (ethylenediaminetetraacetic acid), with the Tris providing buffering capacity to maintain a stable alkaline pH optimal for nucleic acid stability, and EDTA acting as a chelating agent to bind divalent metal ions like Mg²⁺ and Ca²⁺ that are cofactors for degradative enzymes such as nucleases.[2] This combination makes TE buffer essential in protocols involving DNA extraction, purification, and long-term storage, where it prevents enzymatic degradation and supports downstream applications like PCR, cloning, and sequencing.[3][4]In laboratory practice, TE buffer is prepared using molecular biology-grade reagents to avoid contamination, and its pH is critical—typically set between 7.5 and 8.5—to ensure compatibility with restriction enzymes and other molecular tools.[5] Variations exist, such as low-EDTA formulations for applications sensitive to metal chelation, but the standard 10:1 ratio remains the most prevalent due to its balance of protection and minimal interference.[6] TE buffer's role extends beyond storage; it is integral to lysis steps in cell extraction, where it aids in dissolving cellular components while safeguarding genetic material.[1] Its simplicity and effectiveness have made it a staple in both academic research and commercial kits for nucleic acid handling.
Overview
Definition and Purpose
TE buffer is a standard aqueous solution in molecular biology, consisting of Tris (tris(hydroxymethyl)aminomethane) and EDTA (ethylenediaminetetraacetic acid), typically adjusted to pH 8.0, and employed primarily for the handling and preservation of nucleic acids such as DNA and RNA.[7][8] This buffer serves as a versatile medium that supports the solubility of genetic material while minimizing risks of structural damage during laboratory procedures.[9]The primary purpose of TE buffer is to maintain a stable pH environment, which helps prevent the denaturation or hydrolysis of DNA and RNA molecules by counteracting pH fluctuations that could disrupt their phosphodiester backbones.[8] Additionally, the EDTA component acts as a chelating agent, binding divalent metal ions such as magnesium and calcium that are essential cofactors for nuclease enzymes, thereby inhibiting enzymatic degradation of nucleic acids.[1] This dual functionality—pH stabilization and metal ion sequestration—enables TE buffer to solubilize nucleic acids effectively without promoting their breakdown, making it ideal for short- and long-term manipulations.[10]TE buffer has become widely adopted in molecular biology laboratories as a non-toxic and cost-effective solution for the long-term storage and routine handling of genetic material, offering superior stability compared to water or other diluents that may lead to autohydrolysis.[11][8] Its simplicity and reliability have established it as a staple in protocols involving nucleic acid isolation, resuspension, and downstream applications, ensuring the integrity of samples across diverse experimental contexts.[12][13]
Historical Development
The development of TE buffer reflects key advancements in biochemical buffering and chelation techniques during the mid-20th century, aligning with the growth of molecular biology. Tris (tris(hydroxymethyl)aminomethane), a primary buffering agent, was introduced into laboratory practice in the 1940s for maintaining pH in biochemical assays.[14] EDTA (ethylenediaminetetraacetic acid), serving as a metal chelator, was first synthesized in 1935 and adopted in biological applications starting in the 1940s to inhibit enzymes dependent on divalent cations, such as nucleases.[15]The combination of Tris and EDTA to form TE buffer arose in the 1960s and 1970s, driven by the need for stable solutions in early nucleic acid isolation methods. These protocols utilized the buffer to solubilize DNA while preventing degradation through pH control and ion chelation, marking a shift toward more reliable extraction techniques in emerging molecular biology research.[16]TE buffer achieved widespread standardization in the 1970s alongside the advent of recombinant DNA technology, particularly for plasmid isolation from bacteria. Its routine use in lysis and purification steps was codified in influential laboratory manuals, such as the first edition of Molecular Cloning: A Laboratory Manual by Maniatis, Fritsch, and Sambrook (1982), which detailed TE buffer recipes and applications as essential for reproducible nucleic acid handling.In the 1980s, TE buffer's application extended to RNA extraction and storage, leveraging its protective properties to minimize RNase activity during handling. By the 1990s, adaptations like low-EDTA TE buffer (typically 0.1 mM EDTA) emerged to support PCR workflows, reducing interference with magnesium-dependent polymerases while maintaining nuclease inhibition; this variant gained traction in sensitive applications, including early forensic DNA analyses.
Composition and Properties
Chemical Components
The primary component of TE buffer is Tris-HCl, a weak base with the chemical formula C₄H₁₁NO₃·HCl and a molecular weight of 157.60 g/mol.[17] Its pKa value is approximately 8.1 at 25°C, making it suitable for maintaining pH in the range of 7 to 9, which aligns with the stability needs of nucleic acids.[17] In standard TE buffer formulations, Tris-HCl is used at a typical concentration of 10 mM.[18]The second key component is EDTA, ethylenediaminetetraacetic acid, a tetradentate chelating agent with the chemical formula C₁₀H₁₆N₂O₈ for the free acid form, though it is commonly employed as the disodium salt (Na₂EDTA).[19] EDTA exhibits high affinity for divalent metal ions, with stability constants (log K) of approximately 10.7 for Ca²⁺ and 8.7 for Mg²⁺ at 20°C and ionic strength 0.1 M, enabling it to sequester these ions effectively and prevent nuclease activity.[20] The typical concentration of EDTA in TE buffer is 1 mM, often as the disodium dihydrate salt.[18]TE buffer is prepared using ultra-pure water as the solvent, with the pH adjusted to 8.0 using hydrochloric acid (HCl) to optimize the conditions for nucleic acid integrity.[21] This pH value is selected because it falls within the effective buffering range of Tris while promoting the solubility and stability of EDTA.[21]All components must meet stringent purity requirements to avoid contamination; Tris-HCl and EDTA are typically sourced in molecular biology grade, certified DNase- and RNase-free to ensure no degradation of stored nucleic acids.[18] Water used should be nuclease-free, often deionized and treated to remove trace nucleases and metals.[18]
Buffering Mechanism and Stability
The buffering mechanism of TE buffer centers on the Tris-HCl component, which functions through the acid-base equilibrium TrisH⁺ ⇌ Tris + H⁺, with a pKa of 8.1 at 25°C. This equilibrium enables effective resistance to pH fluctuations within the range of 7.5 to 8.5, creating a neutral to mildly alkaline environment that promotes the solubility and structural integrity of nucleic acids such as DNA and RNA.[22][23]Complementing this, EDTA serves as a chelating agent that sequesters divalent cations, including Mg²⁺, which are critical cofactors for endonucleases like DNase I. By binding these ions, EDTA inhibits nuclease activity, thereby enhancing nucleic acid stability; the high stability constant of the EDTA-Mg²⁺ complex (log K = 8.7) ensures robust chelation at the standard 1 mM EDTA concentration.[24]TE buffer's physical properties further support its role in maintaining stable conditions, with low osmolarity that minimizes osmotic effects on biomolecules. The buffer is stable at room temperature for months and can be sterilized by autoclaving or filtration. For long-term storage, TE buffer maintains efficacy when kept at 4°C but may degrade if exposed to contaminating metals or extreme pH shifts, which could compromise its chelating or buffering capacity.[25]
Preparation
Standard Recipe
The standard recipe for preparing 1× TE buffer yields a solution containing 10 mM Tris-HCl and 1 mM EDTA at pH 8.0.[26] Required materials include 1 M Tris-HCl stock solution (pH 8.0), 0.5 M EDTA stock solution (pH 8.0), and ultra-pure water.[27]To prepare 1 L of 1× TE buffer, add 10 mL of 1 M Tris-HCl (pH 8.0) and 2 mL of 0.5 M EDTA (pH 8.0) to approximately 980 mL of ultra-pure water in a suitable container.[28] Mix thoroughly, check and adjust the pH to 8.0 if necessary using HCl or NaOH, then bring the final volume to 1 L with ultra-pure water.[28] Sterilize the solution by filtration through a 0.22 μm filter to ensure sterility without degradation.[29]For a 10× concentrate, which contains 100 mM Tris-HCl and 10 mM EDTA at pH 8.0, combine 100 mL of 1 M Tris-HCl (pH 8.0), 20 mL of 0.5 M EDTA (pH 8.0), and approximately 880 mL of ultra-pure water, then adjust the pH to 8.0 and bring to 1 L as described above before sterilizing by 0.22 μm filtration.[27] Dilute the 10× stock 1:10 with ultra-pure water to obtain working 1× TE buffer.[30]Prepared TE buffer is stable and can be stored at room temperature or 4°C, with labeling of the preparation date and concentration recommended for tracking.[31]
Variant Recipes
Variant recipes of TE buffer modify the standard formulation of 10 mM Tris-HCl and 1 mM EDTA at pH 8.0 to suit specific molecular biology needs, such as reducing interference in enzymatic reactions or enhancing solubility during purification steps.[32]A common variant is low-EDTA TE buffer, which contains 10 mM Tris-HCl and 0.1 mM EDTA (pH 8.0), reducing EDTA concentration by 10-fold to minimize chelation of divalent cations like Mg²⁺ that are essential for enzymes such as Taq polymerase.[33] To prepare 100 mL of low-EDTA TE from stock solutions, dissolve 1 mL of 1 M Tris-HCl (pH 8.0) and 0.02 mL of 0.5 M EDTA (pH 8.0) in approximately 98 mL of ultrapure water, adjust the pH to 8.0 if necessary, and bring the volume to 100 mL with water.[32] This formulation is particularly useful in PCR and forensic short tandem repeat (STR) analysis to prevent inhibition of amplification, as standard EDTA levels can sequester Mg²⁺ and reduce Taq activity.[34]Another variant omits EDTA entirely, using only 10 mM Tris-HCl (pH 8.0) to avoid any metal ion chelation that could impair metal-dependent enzymes, such as certain polymerases or nucleases requiring free Mg²⁺ or other cofactors.[35] Preparation follows the standard method but excludes the EDTA addition, with final pH verification essential to maintain stability.[36]High-salt TE buffer incorporates 100 mM NaCl into the standard recipe (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0) to facilitate DNA precipitation washes or enhance nucleic acid solubility in protocols involving high polysaccharide samples, such as plant extractions. To make 100 mL, add 1 mL of 1 M Tris-HCl (pH 8.0), 0.2 mL of 0.5 M EDTA (pH 8.0), and 2 mL of 5 M NaCl to approximately 95 mL of water, adjust pH, and dilute to volume.[37]All variants undergo the same preparation precautions as the standard TE buffer, including sterilization by 0.22 μm filtration or autoclaving at 121°C for 15 minutes to ensure nuclease-free conditions, followed by pH rechecking post-sterilization due to potential shifts from heat or filtration.[32] These modified recipes are widely available in commercial kits, such as Thermo Fisher's low-EDTA TE or equivalents from suppliers like G-Biosciences, tailored for sensitive downstream assays.[38] The rationale for these adaptations centers on mitigating EDTA's interference in cation-dependent processes while preserving the buffering capacity of Tris.[33]
Applications
Nucleic Acid Storage and Resuspension
TE buffer plays a crucial role in the long-term storage of purified nucleic acids, particularly DNA and RNA, by providing a stable environment that minimizes degradation. Purified DNA is commonly stored at concentrations of 10–100 ng/μL in TE buffer at -20°C, where it exhibits stability for several years without significant loss of integrity. This preservation is attributed to the Tris component maintaining a pH of 8.0, which prevents autocatalytic hydrolysis of the phosphodiester backbone, and the EDTA chelating divalent metal ions such as Mg²⁺ that are essential cofactors for nuclease activity. For RNA, which is more susceptible to degradation, storage in TE at -20°C extends viability for weeks to months, though aliquoting to avoid freeze-thaw cycles is recommended to further inhibit RNase action; for long-term storage of RNA (beyond a few months), -80°C is recommended to maximize stability.[39][40][41][42]Following purification steps like ethanol precipitation, nucleic acids are resuspended in TE buffer to initiate solubilization of the pellet. A typical volume of 20–50 μL of TE buffer is added directly to the dried or air-dried pellet, ensuring the buffer contacts the entire surface by gentle pipetting or vortexing. To enhance dissolution, especially for compacted pellets, the mixture is incubated at 37°C for approximately 10 minutes, allowing complete resuspension without shearing the nucleic acids. This method yields a homogeneous solution ready for storage or immediate use, with the low ionic strength of TE facilitating efficient recovery of 70–90% of the precipitated material.[43][44][8]Compared to nuclease-free water, TE buffer offers distinct advantages for both resuspension and storage by mitigating risks of chemical instability. Pure water can experience pH drops to around 5.0–5.5 due to atmospheric CO₂ absorption, fostering acidic conditions that accelerate depurination—a hydrolytic loss of purine bases that compromises DNA integrity over time. In contrast, TE buffer's buffered pH prevents such shifts, while EDTA actively inhibits residual nucleases by sequestering metal ions, ensuring greater long-term stability even at 4°C for short-term holding.[45][39][42]After resuspension in TE buffer, nucleic acid concentrations are routinely quantified to verify yield and purity prior to storage or downstream applications. Instruments like the NanoDrop spectrophotometer measure absorbance at 260 nm for total nucleic acid content, while the Qubit fluorometer provides dsDNA-specific quantification using intercalating dyes, both performing accurately on samples diluted in TE without interference from the buffer components. These methods allow assessment of concentrations in the 10–100 ng/μL range, confirming suitability for archival storage.[46][47]
Use in Extraction and Purification Protocols
TE buffer plays a crucial role in nucleic acid extraction and purification protocols by stabilizing DNA and RNA during lysis, washing, and elution steps, preventing degradation through its buffering capacity and chelating properties. In traditional organic extraction methods, such as phenol-chloroform, TE buffer is incorporated into lysis solutions to solubilize and protect released nucleic acids from nucleases and pH fluctuations. For instance, in phenol-chloroform protocols, cells are lysed in a buffer containing TE components, followed by phase separation, where TE helps maintain the aqueous phase integrity and facilitates DNA precipitation.[48] Similarly, in CTAB-based extractions for plant tissues, TE buffer is added post-lysis to dissolve the nucleic acid pellet, stabilizing polysaccharides-bound DNA while EDTA inhibits metal-dependent degradative enzymes.[49]During purification, TE buffer is employed in dialysis to remove contaminants like salts and small molecules from crude nucleic acid preparations, ensuring high purity for downstream use. In this process, samples are placed in dialysis tubing submerged in large volumes of TE buffer (e.g., 500 mL), allowing diffusion of impurities while retaining macromolecules over several hours. TE washes are also integral to cesium chloride (CsCl) density gradient centrifugation, where they rinse the gradient interface to collect purified plasmid DNA bands, minimizing ethidium bromide carryover and resuspending pellets in 4 mL TE for further processing.[50]In column-based purification kits, TE buffer serves as the elution medium for silica-membrane spin columns, desorbing high-purity DNA in volumes of 50-100 μL to concentrate yields effectively. For example, in Qiagen QIAprep Spin Miniprep Kits, elution with preheated TE buffer recovers up to 20 μg of supercoiled plasmid DNA from bacterial cultures, with EDTA preventing co-elution of nuclease inhibitors by chelating divalent cations. This step yields DNA at concentrations suitable for immediate use, often 100-500 ng/μL, while maintaining stability during handling.[51][52] In alkaline lysis minipreps, 1x TE is routinely added during the final resuspension to neutralize and stabilize plasmids, typically eluting 5-20 μg from 1-5 mL cultures.[53][1]
Role in Downstream Molecular Analyses
TE buffer plays a critical role in downstream molecular analyses by providing a stable, low-ionic-strength environment for nucleic acids while minimizing interference from its components, particularly EDTA, which can chelate essential divalent cations like Mg²⁺. In polymerase chain reaction (PCR) and quantitative PCR (qPCR), low-EDTA TE buffer (typically 10 mM Tris-HCl, pH 8.0, with 0.1 mM EDTA) is preferred for resuspending or diluting DNA templates to prevent Mg²⁺ chelation that inhibits DNA polymerase activity.[8] Standard TE buffer containing 1 mM EDTA may reduce amplification efficiency by sequestering Mg²⁺, a cofactor required for Taq polymerase, with the dissociation constant (K_d) for Mg²⁺-EDTA binding approximately 10^{-9} M (1 nM) at pH 8.0, leading to suboptimal enzyme kinetics.[54] To mitigate this, DNA is often diluted 1:10 in low-EDTA TE before addition to the master mix, ensuring robust amplification without significant carryover of inhibitory EDTA.[55]In DNA sequencing and cloning workflows, TE buffer is commonly used to resuspend ligation products post-reaction, maintaining nucleic acid integrity during storage or transfer to transformation steps. It is compatible with Sanger sequencing and next-generation sequencing (NGS) library preparation protocols, where the Tris component stabilizes pH and EDTA protects against nuclease degradation.[56] However, if EDTA concentrations exceed 0.1 mM, it can interfere with sequencing enzyme activities by chelating Mg²⁺; in such cases, EDTA is removed using spin columns prior to library prep to avoid reduced read quality or yield.[57]For enzymatic assays, TE buffer serves as a diluent or storage medium, particularly in restriction digests where EDTA is added post-incubation to halt the reaction by inhibiting the Mg²⁺-dependent endonuclease. This prevents non-specific degradation while preserving fragments for downstream applications like gel electrophoresis or cloning. In reporter gene assays, such as those measuring luciferase activity, low-EDTA TE acts as a neutraldiluent for cell lysates or substrates, avoiding pH shifts or cation interference that could affect enzyme kinetics.[58]Key precautions in these analyses include using low-EDTA variants to limit Taq inhibition, as high EDTA (>0.1 mM) can directly bind the polymeraseactive site with a K_d of 47 nM, independent of chelation, potentially reducing activity by over 50% at typical carryover levels.[59] In forensic short tandem repeat (STR) kits, low-TE buffer is specified for DNA extracts to sustain amplification efficiency above 95%, ensuring complete profiles from low-template samples without stochastic effects from Mg²⁺ depletion.[60]