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Universe Hot Start High-Fidelity 2x PCR Master Mix

Description

c. The annealing temperature set up should be based on the Tm of the primers.d. For primers with annealing temperatures ≥ 72°C, a 2-step protocol is recommended.

Biotool Universe Hot Start High-Fidelity 2x PCR Master Mix is a convenient premix version of Universe High-Fidelity Hot Start DNA Polymerase, which is a superior enzyme in this category for robust PCR with extreme fidelity, featuring 53x error rate lower than Taq polymerase and extension rate as fast as 1 sec / kb*. Universe High-Fidelity Hot Start DNA Polymerase possesses 5´→ 3´polymerase activity, 3´ → 5´exonuclease activity and will generate blunt-ended products. The polymerase is also capable of amplifying long fragments such as 20kb genomic DNA and 40kb λDNA. Our suggested applications include cloning, sequencing, genomic DNA amplification and high throughput PCR, etc.* The extension rate varies with individual application.

• General guidelines for low complexity DNA (e.g. plasmid, virus or λDNA) are: 10 pg–30 ng per 50 µl reaction volume; for high complexity genomic DNA, the amount of DNA template should be 50 – 400 ng per 50 µl reaction volume. cDNA synthesis reaction mixture is used as template, the volume should not exceed 10 % of the final PCR reaction volume. Notes: Add the components into sterile PCR tubes while mixing gently.

2). Place PCR tubes to a PCR cycler.3). Perform PCR reaction using optimized cycling conditions. Suggested cycling parameters for using Universe Hot Start DNA Polymerase are provided below. Analyze PCR amplification products on a 0.7–1.0% (w/v) agarose gel.

ContentsCat#:B22101

(100 rxns)Cat#:B22102

(250 rxns)Cat#:B22103

(500 rxns)

Universe Hot Start High-Fidelity

2x PCR Master Mix1.25 mL x 2 1.25 mL x 5 1.25 mL x 10

Segment Number Of Cycles Temperature °C Duration Temperature °C Duration

3-Step Protocol 2-Step Protocol

1

1

1

1.Initial Denaturation

3. Final Extension

4. Hold

2. PCR

95

95

95

95

55-65c

3 min(30 sec)a

15 sec25-35b

7272d

72

4

72

4

15 sec 15 sec

15-30 sec/ kb

3 min(30 sec)a

15-30 sec/ kb

5 min 5 min

∞ ∞

Contents 25 µL Reaction Volume

50 µL Reaction Volume

Universe Hot Start High-Fidelity 2x PCR Master Mix

DNA template (100ng / µL)

Upstream primer (10 µM)

Downstream primer (10 µM)

Distilled water (dH2O)

Total reaction volume

12.5 µL

Variable

25 µL

Variable

1 µL

1 µL

To 25 µL

25 µL

2 µL

2 µL

To 50 µL

50 µL

Components

Notice1. Please make sure the reagent is fully liquid and resuspended prior to use.

Storage• All reagents should be stored at -20 °C

1). All components should be mixed as recommended below prior to use.

Note: a. This mix is based on a hot-start DNA polymerase, the pre-denaturation activation condition should be set to 95°C for 3 minutes (for genomic DNA and cDNA) or 30sec (for plasmid DNA and virus DNA) to thoroughly activate the enzyme.b. Optimized cycling parameters may not necessarily be transferable between thermal cyclers. Consult the instrument manufacturer’s recommendations if further optimization of cycling parameters is required.

Protocol

Order & InquiryTel: (713)732-2181 Fax: +1-866-747-4781E-mail: order@biotool.com

Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: eu.order@biotool.com

Note: a. This mix is based on a hot-start DNA polymerase, the pre-denaturation activation condition should be set to 95°C for 3 minutes (for genomic DNA and cDNA) or 30sec (for plasmid DNA and virus DNA) to thoroughly activate the enzyme.b. Optimized cycling parameters may not necessarily be transferable between thermal cyclers. Consult the instrument manufacturer’s recommendations if further optimization of cycling parameters is required.

• High quality or purified DNA templates are preferred to enhance the success of PCR.• Repeat and make sure that there are no pipetting errors.• Use fresh high quality dNTPs.• Do not use dNTP mix or primers that contain dUTP or dITP.• Sample concentration may be too low. Use more template.• Template DNA may be damaged. Use carefully purified template and make sure template is not fragmented.• Increase extension time.• Increase the number of cycles.• Optimize annealing temperature.• Optimize enzyme concentration.• Optimize the denaturation time.• Check the purity and concentration of the primers.• Check primer design.

• Decrease enzyme concentration.• Decrease extension time.• Reduce the total number of cycles.• Increase annealing temperature or try 2-step protocol.• Vary denaturation temperature.• Decrease primer concentration.

• Increase annealing temperature.• Decrease extension time.• Decrease enzyme concentration.• Titrate template amount.• Decrease primer concentration

No product at all or low yield

Non-specific products - High molecular weight smears

Non-specific products - Low molecular weight discrete bands

Trouble Shooting