Why is DTT used in PCR?

Asked By: Patrina Vosskamp | Last Updated: 22nd February, 2020
Category: science chemistry
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DTT (Dithiothreitol) is a very useful reducing Agent for disulfide bonds. With this it stabilizes enzyms and proteins which posses free sulhydryl groups. DTT breaks di-sulfid bond and loosen the secondary structure of RNA and helps in initiation of transcription so it must for cDNA synthesis.

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Similarly, you may ask, what does DTT do in PCR?

Thermo Scientific DTT (DL-Dithiothreitol; Clelands reagent) is used to stabilize enzymes and other proteins, which possess free sulfhydryl groups.

Beside above, does DTT inhibit PCR? Forensic DNA analysis is partly limited by PCR-inhibitory compounds present in the DNA extracts. We have found that dithiothreitol (DTT) from the DNA extraction process can cause another type of real-time PCR disturbance, i.e., inhibition of signal detection through fluorescence quenching.

Secondly, what is the purpose of DTT?

DTT is used as a reducing or "deprotecting" agent for thiolated DNA. DTT is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins.

What is DTT chemistry?

Dithiothreitol (DTT) is a redox reagent also known as Cleland's reagent. It is used to break down protein disulfide bonds and stabilize enzymes and other proteins. DTT is a small molecule and is an epimeric compound of dithioerythritol (DTE) These reducing reagent products are readily supplied by AG Scientific, Inc.

20 Related Question Answers Found

Why are divalent cations used in PCR?

Magnesium's 2+ charge attracts the electrons of dNTP, allowing the upcoming DNA strand to attack (and attach) the first phosphate group of dNTP. So, divalent cations are needed to keep away the electron charge.

How long does DTT last in solution?

In lyophilized form, the chemical is stable for 12 months. Once in solution, store at -20ºC and use within 3 months to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles. Directions for Use: DTT is supplied as a lyophilized powder.

What is protein reduction?

Reducing agents are used in the reduction of disulfide bonds of proteins and peptides. It is often necessary to remove the reducing agents from the protein/peptide solutions to prevent them from interfering with subsequent procedures.

How do you dissolve a DTT?

Dissolve 1.5 g of DTT in 8 mL of H2O. Adjust the total volume to 10 mL, dispense into 1-mL aliquots, and store them in the dark (wrapped in aluminum foil) at -20°C (indefinitely). Do not autoclave DTT or solutions containing it.

What is the concentration of DTT in loading buffer?

dithiothreitol (DTT or Cleland's reagent) may be used at a final concentration of 350 mM (54 mg/ml).

How long is DTT stable at 4c?

Before bottle opening , the reagent remains stable, protect from light, for 12 months at 2-8 °C, while, after opening it, the stability not is beyond 30 days at 2-8°C, always from light protect.

How do I make 1m DTT?

Preparation of 1 M Dithiothreitol (DTT) Stock Solution
  1. Dissolve 15.45 g of dithiothreitol in 100 mL of water to obtain a final concentration of 1 M (154.5 mg/mL).
  2. Aliquot into 1.6 mL tubes and store the tubes at -20 °C.

How can disulfide bonds be reduced?

Disulfide-reducing reagents are routinely used in biochemical manipulations for (i) reducing the native disulfide bonds in proteins and (ii) maintaining the essential thiol groups in proteins by preventing their oxidation to the disulfide state. Dithiothreitol (DTT) is the most popular disulfide-reducing reagent.

How does TCEP work?

TCEP selectively and completely reduces even the most stable water-soluble alkyl disulfides over a wide pH range. TCEP effectively reduces disulfide bonds over a broad pH range. TCEP is resistant to air oxidation. Compared to DTT, TCEP is more stable, more effective, and able to reduce disulfide bonds at lower pHs.

Why is NaCl used in lysis buffer?

Most lysis buffers contain buffering salts (e.g. Tris-HCl) and ionic salts (e.g. NaCl) to regulate the pH and osmolarity of the lysate. Sometimes detergents (such as Triton X-100 or SDS) are added to break up membrane structures. Lysis buffers can be used on both animal and plant tissue cells.

How long does TCEP last?

TCEP-HCl has been shown to be stable, with 80% of its original reduc- ing ability intact after 21 days at pH values between 1.5 – 11.1. At neutral pH values, phosphate buffered saline and other phosphate containing re- agents can facilitate the oxidation of TCEP-HCl, 50 to 100% oxidation after 72 hours.

Is DTT soluble in water?

This product is soluble in water (50 mg/ml), yielding a clear, colorless solution. DTT is also soluble in ethanol, acetone, ethylate, chloroform, and ether. DTT solutions should be prepared fresh daily.

How do you make DTT?

1 M Dithiothreitol (DTT)
Dissolve 1.5 g of DTT (DL-dithiothreitol, anhydrous m.w.=154.25) in 8 ml of deionized or distilled H2O. Adjust volume to 10 ml, dispense into 1-ml aliquots, and store in the dark (wrapped in aluminum foil) at –20°C (indefi- nitely). Note: Do not autoclave DTT or solutions containing it.

What do reducing agents do to proteins?

Reducing agents disrupt the disulfide bonds. Disulfide bonds are formed by oxidation of sulfhydryl groups on cysteine. Different protein chains or loops within a single chain are held together by the strong covalent disulfide bonds.

What do disulfide bonds do for a protein?

Function of Disulfide Bonds
Disulfide bonds function to stabilize the tertiary and/or quaternary structures of proteins and may be intra-protein (i.e., stabilizing the folding of a single polypeptide chain) or inter-protein (i.e., multi-subunit proteins such as antibodies or the A and B chains of insulin).

How much BSA should I add to PCR?

BSA concentration of 0.01µg/µl to 0.1µg/ µl can be used.