赛默飞taq酶说明书 Thermo Scientific EP0402 Taq DNA Polymera

PRODUCT INFORMATIONTaq DNA Polymerase

(recombinant)

#EP0402

500 U

Lot: __

Expiry Date: __ Concentration:

5 U/µL

Store at -20°C

http://www.77cn.com.cn/onebio

Ordering Information

Taq DNA Polymerase (recombinant)

Component #EP0401 #EP0402 #EP0405 #EP0406

Taq DNA Polymerase, 5 U/µL

100 U

500 U

5 x 500 U 10 x 500 U

10X Taq Buffer with KCl

0.6 mL 2x1.25 mL 10x1.25 mL 20x1.25 mL 10X Taq Buffer with (NH4)2SO4 0.6 mL 2x1.25 mL 10x1.25 mL 20x1.25 mL 25 mM MgCl2

0.6 mL 2x1.25 mL 10x1.25 mL 20x1.25 mL

Taq DNA Polymerase (recombinant), LC

Component

#EP0403 #EP0404 Taq DNA Polymerase, 1 U/µL 100 U 500 U 10X Taq Buffer with KCl 0.6 mL 2x1.25 mL 10X Taq Buffer with (NH4)2SO4 0.6 mL 2x1.25 mL 25 mM MgCl2

0.6 mL

2x1.25 mL

Rev.12

V

Description

Taq DNA Polymerase is a highly thermostable DNA polymerase of the thermophilic bacterium Thermus

aquaticus. The enzyme catalyzes 5’→3’ synthesis of DNA, has no detectable 3’→5’ exonuclease (proofreading) activity and possesses 5’→3’ exonuclease activity. In addition, Taq DNA Polymerase exhibits deoxynucleotidyl transferase activity, which frequently results in the addition of extra adenines at the 3’-end of PCR products.

Recombinant Taq DNA Polymerase is ideal for standard PCR of amplicons 5 kb or shorter.

Applications

Routine PCR amplification of DNA fragments up to 5 kb (1).

Generation of PCR product for TA cloning. DNA labeling (2-4). DNA sequencing (5).

Source

E.coli cells with a cloned pol gene from Thermus aquaticus YT1.

Definition of Activity Unit

One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 70°C. Enzyme activity is assayed in the following mixture: 67 mM Tris-HCl (pH 8.8 at 25°C), 6.7 mM MgCl2,

1 mM 2-mercaptoethanol, 50 mM NaCl, 0.1 mg/mL BSA, 0.75 mM activated salmon milt DNA, 0.2 mM of each dNTP, 0.4 MBq/mL [3H]-dTTP.

Storage Buffer

The enzyme is supplied in: 20 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl,

0.5% (v/v) Nonidet® P40, 0.5% (v/v) Tween® 20 and 50% (v/v) glycerol.

10X Taq Buffer with KCl

100 mM Tris-HCl (pH 8.8 at 25°C), 500 mM KCl, 0.8% (v/v) Nonidet P40.

10X Taq Buffer with (NH4)2SO4

750 mM Tris-HCl (pH 8.8 at 25°C), 200 mM (NH4)2SO4, 0.1% (v/v) Tween 20.

Inhibition and Inactivation

Inhibitors: ionic detergents (deoxycholate, sarkosyl and SDS) at concentrations higher than 0.06, 0.02 and 0.01%, respectively (6).

Inactivated by phenol/chloroform extraction.

PROTOCOL

To prepare several parallel reactions and to minimize the possibility of pipetting errors, prepare a PCR master mix by mixing water, buffer, dNTPs, primers and Taq DNA

Polymerase. Prepare sufficient master mix for the number of reactions plus one extra. Aliquot the master mix into inpidual PCR tubes and then add template DNA. 1. Gently vortex and briefly centrifuge all solutions after thawing.

2. Place a thin-walled PCR tube on ice and add the following components for each 50 µL reaction: 10X Taq Buffer 5 µL dNTP Mix, 2 mM each (#R0241) 5 µL (0.2 mM of each)Forward primer 0.1-1.0 µM Reverse primer 0.1-1.0 µM 25 mM MgCl2* 1-4 mM Template DNA 10 pg - 1 µg Taq DNA Polymerase 1.25 U Water, nuclease-free (#R0581)

to 50 µL

Total volume 50 µL

*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:

Final concentration of MgCl2, mM 1 1.5 2 2.5 3 4 Volume of 25 mM MgCl2 to be added for 50 µL reaction, µL

2

3

4

5

6

8

3. Gently vortex the samples and spin down.

4. If using a thermal cycler that does not use a heated lid, overlay the reaction mixture with 25 µL of mineral oil. 5. Perform PCR using recommended thermal cycling conditions:

Step °C Time

Number of

cycles Initial denaturation 95 1-3 min 1

Denaturation 95 30 s Annealing Tm-5 30 s 25-40 Extension

72 1 min/kb Final Extension 72

5-15 min

1

GUIDELINES FOR PREVENTING CONTAMINATION

OF PCR REACTION

During PCR more than 10 million copies of template DNA are generated. Therefore, care must be taken to avoid contamination with other templates and amplicons that may be present in the laboratory environment. General

recommendations to lower the risk of contamination are as follows:

Prepare your DNA sample, set up the PCR mixture, perform thermal cycling and analyze PCR products in separate areas.

Set up PCR mixtures in a laminar flow cabinet equipped with an UV lamp.

Wear fresh gloves for DNA purification and reaction set up.

Use reagent containers dedicated for PCR. Use positive displacement pipettes, or use pipette tips with aerosol filters to prepare DNA samples and perform PCR set up. Use PCR-certified reagents, including high quality water (e.g., Water, nuclease-free, (#R0581)).

Always perform “no template control” (NTC) reactions to check for contamination.

GUIDELINES FOR PRIMER DESIGN

Use the ThermoScientific REviewer primer design software recommendations for PCR primer design as outlined below: PCR primers are generally 15-30 nucleotides long. Optimal GC content of the primer is 40-60%. Ideally, C and G nucleotides should be distributed uniformly along the primer.

Avoid placing more than three G or C nucleotides at the 3’-end to lower the risk of non-specific priming. If possible, the primer should terminate with a G or C at the 3’-end.

Avoid self-complementary primer regions,

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