1. Home
  2. DNA polymerases and buffers

DNA polymerases and buffers

Introduction

Polymerase chain reaction (PCR) is powerful technique used for in vitro exponential amplification of specific DNA region (target region) that lies between two regions of known DNA sequence (oligonucleotide primer binding sites).

There are three major steps in PCR performed at different temperatures. The first is denaturation of template DNA at 94oC in which double-stranded DNA melts to single-stranded DNA, and all enzymatic reactions stop. Next is annealing step (at 50 - 68oC, depending on primers composition) in which oligonucleotide primers bind to the template. Final step is extension in which DNA complementary to template DNA is synthesized at direction 5´->3´ at 72oC. These three steps are repeated (usually 20 - 40 times) and in each step the amount of the amplified DNA fragment is doubled.

Reaction mixture for PCR contains the following components: thermostable DNA polymerase, reaction buffer with Mg2+, deoxynucleoside triphosphates set (dATP, dGTP, dCTP and dTTP), template DNA and a pair of oligonucleotide primers (usually 20 - 30 nucleotides in specific sequence). Top-Bio offers six products containing thermostable DNA polymerases extensively tested for their suitability for PCR. These enzyme preparations differ in concentration, composition of storage buffers and presence of additives. 

Taq DNA polymerase

  • This product contains Taq DNA polymerase at a concentration of 5 U/μl in storage buffer and is widely used for routine diagnosis purposes.

Taq DNA polymerase Unis

  • This product contains Taq DNA polymerase at a concentration of 5 U/μl in optimized storage buffer which increases stability of the enzyme and robustness of PCR

Taq-Purple DNA polymerase

  • In this product, concentration of Taq DNA polymerase is reduced to 1 U/μl and storage buffer is supplemented with inert Dye for easy loading and control of the presence of the enzyme in PCR.

Taq DNA polymerase 1.1

  • This product contains Taq DNA polymerase at a concentration 1 U/μl for easier manipulation. Furthermore, it is supplied with modified buffer (Blue buffer) which is more tolerant to elevated Mg2+ concentrations and decreases production of nonspecific products.

LA DNA Polymerases Mix

  • This product contains two DNA polymerases at a concentration of 5 U/μl. One of them, Taq DNA polymerase, facilitates rapid and robust synthesis of DNA, whereas the second one with 3´->5´ exonuclease activity enhances PCR fidelity (~5 times higher when compared with Taq DNA polymerase). LA DNA polymerases Mix is suitable for amplification of long DNA fragments (up to 40 kbp) and also for amplification of DNA fragments aimed for DNA cloning. 

10x concentrated PCR Blue buffer

  • PCR Blue Buffer contains ammonium sulfate instead of potassium chloride and thereby exhibits higher tolerance towards changing concentrations of MgCl2.
Sort products
10x conc. PCR Blue Buffer without MgCl2+MgCl2
PCR Blue Buffer contains ammonium sulfate instead of potassium chloride and thereby  exhibits higher tolerance towards changing concentrations of MgCl2. The reaction buffer can be used with the following Top-Bio polymerases: Taq DNA polymerase (Cat. No T032-T034), Taq DNA polymerase Unis (Cat. No. T037-T039), Taq-Purple DNA polymerase (Cat. No T107-T109), Taq DNA polymerase 1.1 (Cat. No T112-T114) and Combi Taq DNA polymerase (Cat. No C205-C207). 1x concentrated buffer contains 2.5 mM MgCl2, which is suitable for most PCRs. Alternatively, 10x concentrated buffer is provided without MgCl2 and 25 mM MgCl2 is provided separately. This allows rapid optimization of reaction conditions.
LA DNA Polymerases Mix
LA DNA polymerases Mix is unique blend of two thermostable DNA polymerases. One of them is Taq DNA polymerase which is highly processive but lacks a 3’->5’ exonuclease proofreading activity, which accounts for relatively high error rate during DNA amplification. Second polymerase is less processive but possesses 3’->5’ exonuclease activity necessary for proofreading. The polymerases mix decreases error rate during synthesis of complementary DNA strands and preserves high speed of DNA synthesis.
Taq DNA polymerase 1.1
This product is a modification of Taq DNA polymerase Unis (Cat. No. T 037-T 039). Difference between these two products rely in lower concentration of the polymerase (1 U/µl) , which makes easier pipetting of the enzyme. Furthermore, the enzyme is supplied with 10x concentrated PCR Blue buffer. This buffer contains ammonium sulfate, which contributes to broader tolerance of MgCl2 and lower production of nonspecific enzymes.
Taq DNA polymerase
Taq DNA polymerase is a thermostable enzyme isolated from Thermus aquaticus. The enzyme catalyzes synthesis of complementary DNA strand in the 5’->3’ direction and also possesses a 5’->3’ exonuclease activity. During amplification of DNA fragments, Taq polymerase adds at 3’ end an adenosine overhang. This can be utilized for cloning of PCR-generated DNA fragments. Advantage of the enzyme is its high processivity [amplification of 1000 base pairs (bps) takes < 1 min]. Disadvantage of the enzyme is that it lacks a 3’->5’ exonuclease proofreading activity and this accounts for high error rate (about 1 error to 105 - 106 base bps). The major usage of the enzyme is in diagnostic analysis  for amplification of DNA fragments up to 5000 bps.
Taq DNA polymerase Unis
Taq DNA polymerase Unis is an alternative to Taq DNA polymerase (Cat. No. T032-T034). Difference between these two products is in difference in enzyme storage buffer which enhances stability and efficiency of the enzyme. Occasional amplification of nonspecific fragments can be prevented by enhanced dilution of the enzyme.
Taq-Purple DNA polymerase
Taq-Purple DNA polymerase is an alternative to Taq DNA polymerase Unis (Cat. No. T037-T 039). In this product, concentration of Taq DNA polymerase is reduced to 1U/μl and storage buffer is supplemented with inert Dye for easy loading and control of the presence of the enzyme in PCR.
Q-Taq DNA polymeráza