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 In certain countries, patents cover the PCR process. This product is intended for researchers having a license to perform PCR or those not required to obtain a license.

UptiPfu DNA Polymerase

Recombinant DNA Polymerase with 5’-3’ polymerase activity and 3’-5’ exonuclease proofreading activity suitable for: Amplifications requiring high fidelity synthesis Amplifications involving a high number of cycles (up to 55-60) Amplifications up to 5kbp Amplifications involving genomic DNA Amplifications with limiting amounts of template Multiplex amplifications…

UptiPfu DNA Polymerase increases the sensitivity of the reaction!!! Error rate: around 1 x 10-6, 10-fold lower than non-proofreading enzymes Highly thermostable Higher Fidelity (1/10 Kbp)  than Taq (1/6 Kbp) Higher Amplification Yields High Sensitivity and Reproducibility Amplification products can be directly used in blunt-ended vectors ... and much much more!

UptiPfu DNA Polymerase,  1 U/ µl Cat.# Product UPAK5100 100 units + Standard buffer UPAK5101 250 units + Standard buffer UPAK5102 500 units + Standard buffer UPAK5103 100 units + MgCl2 free buffer UPAK5104 250 units + MgCl2 free buffer UPAK5105 500 units + MgCl2 free buffer

Nucleotides

Nucleotides, UltraPure

Nucleotides solutions, Sets

Nucleotides solutions, Mix

Desoxy Nucleotides

Nucleotides, UltraPure dATP-Na4 dGTP-Na4 dCTP-Na4 dUTP-Na4 dITP-Na4

dATP-Na4 UP314671 25µol (25 µmol in solution 100 mM, pH 7.0) 2´ -Deoxyadenosine 5´ -triphosphate, tetrasodium salt C10H12N5O12P3Na4 CAS [1927-31-7] MW 579.13 Absorbance max.: 259nm (pH 7) e at at absorbance max.: 15 E x mmol-1x cm-1 Purity : > 98 % Applications : dATP is used as a substrate for sequencing DNA by the method of Sanger.1.2 DATP is used as a substrate for PCR.3.4 Literature : 1. Burd, J.F., Wells, R.D., J.Mol.Biol., 53 :435 (1970) 2. Sanger, F. et al., Proc. Natl. Acad. Sci. USA, 74 :5463 (1977) 3. Mullis, K.B. and Faloona, F.A., Methods Enzymol. 155 :335 (1987) 4. Newton, C.R. and Graham, A., PCR Spectrum Akad. Verlag Heidelberg (1997)	dGTP-Na4 UP982151 25µmol (25 µmol in solution 100 mM, pH 7.0) 2´-Deoxyguanosine 5´-triphosphate, tetrasodium salt C10H12N5O13P3Na4 CAS [93919-41-6] MW 595.18 Absorbance max. : 252 nm (pH 7) e at absorbance max.:13.7 E x mmol-1 x cm-1 Purity : > 98 %
dCTP-Na4 UP314781 25µmol (25 µmol in solution 100 mM, pH 7.0) 2´- Deoxycytidine 5´ -triphosphate, tetrasodium salt Absorbance max. : 267 nm (pH 7) e at absorbance max.: 9.6 E x mmol-1x cm-1 Purity : > 98 %	dUTP-Na4 UP636831 25µmol (25 µmol in solution 100 mM, pH 7.0) 2´-Deoxyuridine 5´-triphosphate, tetrasodium salt C9H11N2O14P3Na4 CAS [102814-08-4] MW 556.10 Absorbance max. : 262  nm (pH 7) e at absorbance max.: 10.2 E x mmol-1x cm-1 Purity : > 98 %
dITP-Na4 UP637564 25µmol (25 µmol in solution 100 mM, pH 7.0) 2´ - Deoxyinosine 5´ -triphosphate, tetrasodium salt C10H11N4O13P3Na4 CAS [95648-77-4] MW 580.10 Absorbance max. : 249 nm (pH 7) e at absorbance max.: 12.2 E x mmol-1x cm-1 Purity : > 98 %	All nucleotides are stored at -20°C

Nucleotides solutions, Sets

Uptima dNTPs solutions are prepared with UltraPure nucleotides in reproducible sets (100µM concentration), and in ready to use mixtures (at 10mM and 2mM concentrations). Store at -20°C dNTP Set1 dNTP Set2

-	Formula	Molecular Weight	l max (pH 7.0)	e at l max	PH of solution	Purity
dATP-Na4	C10H12N5O12P3Na4	579.13 g/mol	259 nm	15.4 E x mmmol-1 x cm-1	7.0	> 98%
dGTP-Na4	C10H12N5O13P3Na4	595.18 g/mol	252 nm	13.7 E x mmmol-1 x cm-1	7.0	> 98%
dCTP-Na4	C9H12N3O12P3Na4	555.11 g/mol	272 nm	9.1 E x mmmol-1 x cm-1	7.0	> 98%
dTTP-Na4	C10H13N2O14P3Na4	570.10 g/mol	267 nm	9.6 E x mmmol-1 x cm-1	7.0	> 98%
-
dATP-Na4	C10H12N5O12P3Na4	579.13 g/mol	259 nm	15.4 E x mmmol-1 x cm-1	7.0	> 98%
dGTP-Na4	C10H12N5O13P3Na4	595.18 g/mol	252 nm	13.7 E x mmmol-1 x cm-1	7.0	> 98%
dCTP-Na4	C9H12N3O12P3Na4	555.11 g/mol	272 nm	9.1 E x mmmol-1 x cm-1	7.0	> 98%
dUTP-Na4	C9H11N2O14P3Na4	556.10 g/mol	262 nm	10.2 E x mmmol-1 x cm-1	7.0	> 98%

DNTP Set1 UP968640 4x25µmol (100mM of dATP, dGTP, dCTP, dTTP) UP968641 20x25µmol dNTPs are used as substrates for sequencing DNA by method of Sanger(1,2). dNTPs are used as substrates for PCR(3,4). Technical Sheet Literature : (1) Sanger, F., Nicklen, S., and Coulson, A.R, Proc.Natl.Acad. Sci. USA, 74, 5463 (1977) (2) Biggin, M.D., Gibson, T.G., and Hong, G.F., Proc.Natl.Acad.Sci. USA, 80, 3963 (1983) (3) Mullis, K.B., and Faloona, F.A., Methods Enzymol. 155, 335 (1987) (4) Newton, C.R., and Graham, A., PCR Spectrum Akad. Verlag Heidelberg (1997).

DNTP Set2 UP968660 4x25µmol (100mM of dATP, dGTP, dCTP, dUTP) UP968661 20x25µmo dNTPs are used as substrates for PCR(1,2). Literature : (1) Mullis, K.B., and Faloona, F.A., Methods Enzymol. 155, 335 (1987) (2) Newton, C.R., and Graham, A., PCR Spectrum Akad. Verlag Heidelberg (1997).

Nucleotides solutions, Mix Mix PCR 1 Mix PCR 2 Mix PCR 3 Mix PCR4 Mix Seq T

Mix PCR 1 UP926890 1ml (2mM of dATP, dGTP, dCTP, dTTP) UP926891 5x1ml Ready-to-use dNTP mixture for: UptiTherm DNA Polymerase Taq DNA polymerase (1). PH: 7.0 Purity >98% Literature : (1) De Noronha, C., and Mullins, J., PCR Methods and applications 2, 131-136 (1992) Mix PCR 3 UP984440 0.2ml (10mM of dATP, dGTP, dCTP, dTTP) UP984441 5x0.2ml Ready-to-use dNTP mixture for: UptiTherm DNA Polymerase Taq DNA polymerase (1). PH: 7.0 Purity >98% Mix Seq T - Termination mixture UP996780 4x0.3ml Ready-to-use dNTP/ddNTP Set for UptiTherm DNA Polymerase and Taq DNA Polymerase Contains: 20µM dATP, dCTP, dGTP, dTTP (of each), 350 µM ddATP UP984470 0.3ml 20µM dATP, dCTP, dGTP, dTTP (of each), 350 µM ddTTP UP984510 0.3ml 20µM dATP, dCTP, dGTP, dTTP (of each), 350 µM ddGTP UP984520 0.3ml 20µM dATP, dCTP, dGTP, dTTP (of each), 350 µM ddCTP UP984530 0.3ml Purity : > 98% PH : 7.0 Applications: Termination mixture ddATP, ddTTP, ddGTP, and ddCTP are provided in four differents coloured vials respectively as : red, green, yellow and blue.

Mix PCR 2  UP982160 1ml (2mM of dATP, dGTP, dCTP, dUTP) UP982161 5x1ml Ready-to-use dNTP mixture for: UptiTherm DNA Polymerase Taq DNA polymerase (1). PH: 7.0 Purity >98% - - - Mix PCR 4 UP984450 0.2ml (10mM of dATP, dGTP, dCTP, dUTP) UP984451 5x0.2ml Ready-to-use dNTP mixture for: UptiTherm DNA Polymerase Taq DNA polymerase (1). PH: 7.0 Purity >98% - - -

Desoxy nucleotides ddATP ddGTP ddCTP ddTTP ddNTP Set

ddATP UP638400 1µmol ddATP-Li4 2´, 3´ -Dideoxyadenosine 5' -triphosphate, tetralithium salt C10H12N5O11P3Li4 MW 498.93 Solution at 10 mM, pH 7.0 absorbance max.: 260 nm (pH 7) e at absorbance max.: 15.3 E x mmol-1 x cm-1 Purity : > 98 % Applications: ddATP is used as a substrate for sequencing DNA by the method of Sanger. -A ddCTP UP637520 1µmol ddCTP-Li4 2´, 3´ -Dideoxycytidine 5' -triphosphate, tetralithium salt C9H12N3O12P3Li4 MW 474.90 Solution at 10 mM, pH 7.0 absorbance max.: 252 nm (pH 7) e at absorbance max.: 13.7 E x mmol-1 x cm-1 Purity : > 98 % Applications: Incorporation of ddGTP by DNA polymerases onto the 3’-hydroxyl of the extending transcript inhibits further extension. This principle is used in DNA sequencing.

ddGTP UP638000 1µmol ddGTP-Li4 2´, 3´ -Dideoxyadenosine 5' -triphosphate, tetralithium salt C10H12N5O12P3Li4 MW 514.93 Solution at 10 mM, pH 7.0 absorbance max.: 252 nm (pH 7) e at absorbance max.: 13.7 E x mmol-1 x cm-1 Purity : > 98 % Applications: Incorporation of ddGTP by DNA polymerases onto the 3’-hydroxyl of the extending transcript inhibits further extension. This principle is used in DNA sequencing. ddTTP UPA13250 1µmol ddTTP-Li4 2´, 3´ -Dideoxythymidine 5' -triphosphate, tetralithium salt C10H13N2O13P3Li4 MW 489.91 Solution at 10 mM, pH 7.0 absorbance max.: 267 nm (pH 7) e at absorbance max.: 9.5 E x mmol-1 x cm-1 Purity : > 98 % Applications: Incorporation of ddGTP by DNA polymerases onto the 3’-hydroxyl of the extending transcript inhibits further extension. This principle is used in DNA sequencing.

ddNTP Set

-

UP968620

4x1µmol

-

-

Store at -20°C

DdNTPs are used as substrates for sequencing DNA by method of Sanger^(1,2).

Literature :

(1) Sanger, F., Nicklen, S., and Coulson, A.R, Proc.Natl.Acad. Sci. USA, 74, 5463 (1977)

(2) Biggin, M.D., Gibson, T.G., and Hong, G.F., Proc.Natl.Acad.Sci. USA, 80, 3963 (1983)

ddATP-Li₄

ddGTP-Li₄

ddCTP-Li₄

ddTTP-Li₄

Formula

C₁₀H₁₂N₅O₁₁P₃Li₄

C₁₀H₁₂N₅O₁₂P₃Li₄

C₉H₁₂N₃O₁₂P₃Li₄

C₁₀H₁₃N₂O₁₃P₃Li₄

Molecular Weight

498.93 g/mol

514.93 g/mol

474.90 g/mol

489.91 g/mol

l max (pH 7.0)

260 nm

252 nm

272 nm

267 nm

e at l max

15.3 E x mmmol^-1 x cm^-1

13.7 E x mmmol^-1 x cm^-1

9.6 E x mmmol^-1 x cm^-1

9.5 E x mmmol^-1 x cm^-1

Concentration

10 mM

10 mM

10 mM

10 mM

Amount

1 µmol

1 µmol

1 µmol

1 µmol

PH of solution

7.0

7.0

7.0

7.0

Purity

> 98%

> 98%

> 98%

> 98%

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