Molecular Biology

dna amplification

DNA polymerase - Nucleotides

• Universal Real Time PCR

• DNA Polymerase

  • UptiTherm DNA Polymerase - recombinant, from thermus thermophilus

    UptiTherm DNA Polymerase - in solution - Gel form UptiTherm DNA Polymerase - UptiTherm EC DNA Polymerase

    Related Product: UptiStart

  • UptiTherm Hot Start PCR Master Mix

  • UptiPfu DNA Polymerase - recombinant, with 5’-3’ polymerase activity and 3’-5’ exonuclease proofreading activity

    UptiPfu DNA Polymerase

• Nucleotides

  • Nucleotides, UltraPure:

    dATP-Na_{4 -} dGTP-Na_{4 -} dCTP-Na_{4 -} dUTP-Na_{4 -} dITP-Na₄

  • Nucleotides solutions, Sets:

    dNTP Set1 - dNTP Set2

  • Nucleotides solutions, Mix:

    Mix PCR 1 - Mix PCR 2 - Mix PCR 3 - Mix PCR4 - Mix Seq T

  • Desoxy nucleotides:

    ddATP - ddGTP - ddCTP - ddTTP - ddNTP Set

Uptima Universal Real Time PCR Master Mix Kit

Description

The Uptima Universal Real-Time PCR Master Mix is a single-tube 2X reagent ideal for most quantitative real time PCR applications. The Uptima Real Time PCR kit support quantitative amplification and detection with multiplex capability and show consistent high performance with various fluorescent detection systems including Green DNA I detection, TaqMan probes and Molecular beacons. The Uptima Real Time PCR kit has been designed for optimal performance on ABI PRISMTM Instruments, the LightCyclerTM Instrument, the Mx4000TM Instrument and the DNA Engine OpticonTM System.  The Uptima Real Time PCR kit includes the components necessary for performing PCR amplification, and have been successfully used for amplify and detect a variety of DNA target such as genomic DNA, cDNA and plasmid DNA.

To prevent template cross-contamination the master mix are design for future UNG (Uracil-N-glycosylase) treatment by replacement of dTTP with dUTP, however the master mix does not contain the UNG enzyme in present kit version.

Key Features

• Contain TEMPase enzyme for increased specificity and product yield

• Detection of low target copy number

• Designed to diminish the formation of non-specific product

• 2x Master Mix in one tube. Add primers, probe and template

The kit contains:

• 2x Uptima Real Time PCR Master Mix, Reference Dye, Green Dye I, MgCl2 and glass Blocking Agents.

• Sufficient for 200 reactions of 50 µl pr. reaction

Technical Sheet

Construction of standard plasmids for SYBR green quantitative PCR.

UptiTherm DNA Polymerase - recombinant, from thermus thermophilus

• Works at elevated temperatures

• Lower error rate than Taq polymerase

• Efficient for long range applications

Applications/Benefits

Source and description

For amplifications involving sequences homologous to E. coli

Figure1.

Amplification performance for long range applications.

Two DNA fragments of 3.5 and 5Kbp were amplified using UptiTherm DNA Polymerase for the amplification reaction, in a reaction volume of 50µl, including 10 pmoles of each primer in the supplied buffer, 200µM dNTP (Cat.No. UPS54211) and three (3) units of DNA Polymerase.

The amplification was performed after a previous phase of denaturation of 5 min at 94ºC, 35 cycles of amplification (30 seconds at 94ºC, 30 seconds at 55ºC, 3 minutes at 72ºC) and a final extension of 7 minutes at 72ºC.

5µl of each amplification reaction were loaded in a 0.8% agarose MB gel (Cat.No. UPS54171).

Quality control

Users may be required to obtain a license depending on the country and/or application.

UptiTherm DNA Polymerase - in solution

Gel form UptiTherm DNA Polymerase

UptiTherm EC DNA Polymerase

UptiTherm DNA Polymerase - in solution

• The concentration of 1U/µl has been designed for ease of use, and in order to avoid pipetting errors. It’s use helps prevent inhibition/inespecificity of the reaction due to excess enzyme.

• The ‘traditional’ concentration of 5 U/µl suits applications where reaction volumes are critical.

UptiTherm Hot Start PCR Master Mix

Description	10X Uptima Hot Start Buffer (MgCl2 15mM)	MgCl2 50 mM	Size /units	Cat.#
UptiTherm Hot Start PCR Master Mix  With 10X Uptima Hot Start Buffer (MgCl2 15 mM)	1.5 mL	1.5 mL	250	UPQ6587A
	2 x 1.5 mL	2 x 1.5 mL	1000	UPQ6587B
Store at -20°C.  FOR RESEARCH USE ONLY

General Description The Uptima Hot start PCR Kit is ideal for performing automated Hot Start PCR, a modification of the PCR process in which amplification reactions are initiated at an elevated temperature. During Hot Start, primers bind only to their specific target, and polymerase activity is directed exclusively to that target. As a result, only the region of interest is amplified, which increases sensitivity and yield while reducing non-specific background amplification. Uptima Hot start DNA Polymerase is essential for Hot Start PCR. A chemically modified form of the DNA Polymerase, facilitates the Hot Start process by means of its thermal activation property at a temperature well above optimal annealing. The enzyme remains inactive until the time, temperature, and pH are optimal. This improves specificity by prohibiting mis-priming and extension, thus eliminating waste and increasing yield. Because Uptima Hot start DNA Polymerase is a chemical Hot Start enzyme, there is no additional biological contamination. The Uptima Hot start DNA Polymerase also enables an unparalleled level of sensitivity. Sensitivity improves multiplex PCR, an applied PCR technique that amplifies several specific targets simultaneously. Applications that previously required two or more reactions can be performed in a single reaction tube. Hence, multiplexing represents a substantial savings of time and costly reagents.

Thaw 10X Uptima Hot Start Buffer, dNTP mix, primer solutions. It is important to mix the solutions completely before use to avoid localized concentrations of salts. Prepare a master mix according to Table 1. The master mix typically contains all the components needed for extension except the template DNA. Mix the master mix thoroughly and dispense appropriate volumes into reaction tubes. Mix gently, e.g., by pipetting the master mix up and down a few times. Add template DNA to the individual tubes containing the master mix. Program the thermal cycler according to the manufacturer’s instructions. Each program must start with an initial heat activation step at 95°C for 15 minutes. For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair. Place the tubes in the thermal cycler and start the reaction.

Table 1.  Reaction components (master mix and template DNA) Component Vol./reaction Final Conc. 10X Uptima Hot Start Buffer 5 µL 1X dNTP mix (12.5 mM of each) 0.8 µL 0.2 mM of each dNTP Primer A Variable 0.1–1.0 µM Primer B Variable 0.1–1.0 µM UptiTherm Hot Start DNA Polymerase 1 µL 5 units Distilled Water Variable - - - - Template DNA Variable Variable TOTAL volume 50 µL - - - -   Table 2.  MgCl2 concentration in a 50 mL reaction Final MgCl2 conc. in reaction (mM) 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Additional volume of 25 mM MgCl2 per reaction (µL) 0 1 2 3 4 5 6

Key Features  Automated UptiTherm Hot Start enzyme for increased specificity and product yield Successful multiplex reactions saves time and reagents Designed to diminish the formation of non-specific product Detection of low target copy number 10X Uptima Hot Start Reaction Buffer Combination of KCl and (NH4)2S04, 15 mM MgCl2, 1% Tween 20Ò. Uptima Hot Start Storage Buffer Enzyme is supplied in 20 mM Tris-HCl pH 8.3, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% NP40, 50% glycerol. Unit Definition One unit is defined as the amount that incorporates 10 nmoles of dNTPs into acid-precipitable form in 30 minutes at 72°C under standard assay conditions. Quality Control Endonuclease, exonuclease and priming activities are not detected after 3 hours incubation of 1 mg of pUC19 plasmid DNA and 0.5 µg EcoR I digested lambda phage DNA at 72°C in the presence of 40 units of Uptima Hot Start DNA Polymerase. Suggested Protocol using UptiTherm Hot Start DNA Polymerase This protocol serves as a guideline for primer extensions. Optimal reaction conditions such as incubation times, temperatures, and amount of template DNA may vary and must be individually determined. Notes: Set up reaction mixtures in an area separate from that used for DNA preparation or product analysis. 15 mM MgCl2 is present in the 10X Uptima Hot Start buffer. The 1X concentration is 1.5 mM MgCl2. In some applications, more than 1.5 mM MgCl2 is needed for the best results. For this reason, 25 mM MgCl2 is included with the kit. Table 2 provides the volume of 25 mM MgCl2 to add to the master mix if a higher MgCl2 concentration is required.

Related Products Description Cat.# UptiTherm DNA Polymerase (1000 Units) with Mg free Buffer and 50 mM MgCl2 Buffer UPS53921 UptiTherm DNA Polymerase (1000 Units) with Mg free Buffer and 50 mM MgCl2 Buffer and dNTP mix UPS53823 dNTP Mix PCR 3 (0,2 ml at 10  mM of each dA, dC, dG and dT) UP984440 Tween 20Ò is a registered trademark of ICI Americas, Inc. NOTICE