Genie Fusion Ultra High-Fidelity Green 2X Master Mix

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SKU:
MORV0007
€40

Description

Product Information

Genie Fusion Ultra High-Fidelity DNA Polymerase is a new generation superior enzyme based on Genie Fusion Ultra DNA Polymerase for robust PCR with higher fidelity. The unique extension factor, specificity-promoting factors and plateau-inhibiting factor newly added to Genie Fusion Ultra greatly improve its long-fragment amplification ability, specificity, and PCR yield. Genie Fusion Ultra is capable of amplifying long fragments such as 40 kb λ DNA, 40 kb plasmid DNA, 20 kb genomic DNA and 10 kb cDNA. The amplification error rate of Genie Fusion Ultra is 53-fold lower than that of conventional Taq and 6-fold lower than that of Pfu. In addition, Genie Fusion Ultra has a good resistance to PCR inhibitors and can be used for direct PCR amplifications of bacteria, fungi, plant tissues, animal tissues, and even whole blood samples. Genie Fusion Ultra contains two monoclonal antibodies inhibiting the 5´→3´ polymerase activity and 3´→5´ exonuclease activity at room temperature, which enable Genie Fusion Ultra to perform greatly specific Hot-Start PCRs. Genie Fusion Ultra High-Fidelity Green 2X Master Mix contains Genie Fusion Ultra High-Fidelity DNA Polymerase, dNTP, an optimized buffer system, and loading dye. The amplification can start only with the addition of primer and template, thereby easing PCR setup and improving reproducibility. Protective agents in the Genie Fusion Ultra High-Fidelity Green 2X Master Mix enable the resistance to repeated freeze-thaw cycles. Amplification will generate blunt-ended products, which are compatible with GenieClone II One Step Cloning Kit Series (Assay Genie. #MORV0098, #MORV0100, #MORV0102).

Product Components

Components MORV0007 MORV0008 MORV0009

Genie Fusion Ultra High-Fidelity Green 2X Master Mix

1 ml

5 x 1ml

15 x 1 ml

Storage

Store at -20℃; avoid repeated freezing and thawing.

Unit Definition

One unit (U) is defined as the amount of enzyme that incorporates 10 nmol of whole dNTPs into acid-insoluble products in 30 minutes at 74℃ with activated salmon sperm DNA as the template /primer.

Quality Control

Residual Endonulease Test: The product is tested in a reaction containing 25 μl of Genie Fusion Ultra High-Fidelity Green 2X Master Mix and 0.3 μg of Supercoiled pBR322 DNA. After incubation at 37℃ for 4 hours, there is no visually discernible change in DNA bands determined by agarose gel electrophoresis.

ResidualE.Coli gDNA Test: The residual nucleotide in 25 μl of Genie Fusion Ultra High-Fidelity Green 2X Master Mix is tested by SYBR® Green qPCR using specific primers of E.Coli gDNA. The residual E.Coli gDNA is lower than 10 copies.

Functional Assay 1: In a 50μl PCR system with 1U of Genie Fusion Ultra High-Fidelity Green 2X Master Mix, 100 ng of human genomic DNA was used as template. After 35 cycles, 1/10 of PCR products were detected by 1% agarose gel electrophoresis. A single DNA band of 8.2 kb was detected after EB staining.

Functional Assay 2: In a 50μl PCR system with 1U of Genie Fusion Ultra High-Fidelity Green 2X Master Mix, 10 ng of λ DNA was used as template. After 30 cycles, 1/10 of PCR products were detected by 1% agarose gel electrophoresis. A single DNA band of 15 kb was detected after EB staining.

Experimental Process

6.1 For Conventional PCR

Recommended PCR System
Keep all components on ice during the experiment. All components need to be mixed up thoroughly after thawing and put back to -20℃ Immediately for storage after using.

DdH2O

up to 50 μl

Genie Fusion Ultra High-Fidelity Green 2X Master Mix

25 μl

rimer 1 (10 μM)

5 μl

Primer 2 (10 μM)

5 μl

Template DNAª

x μl

The PCR Enhancer (Assay Genie. #MORV0084) is recommended for unsuccessful amplification of fragments with GC content > 60%. a. Optimal reaction concentration varies in different templates. In a 50 µl system, the recommended template usage is as follows:

Templates

Input Template DNA

Genomic DNA

50 - 400 ng

Plasmid or Virus DNA

10 pg- 30 ng

cDNA

1 - 5 μl (≤ 1/10 of the total volume of PCR system)

Recommended PCR Program

Steps

Temperature

Time

Cycles

Pre-denaturation a

95℃

30 sec / 3 min

1

Denaturation

95℃

15 sec

} 25 - 35

Annealing b

56-72℃

15 sec

Extension c

72℃

30 - 60 sec / kb

Final Extension

72℃

5 min

1

a. For pre-denaturation, the recommended temperature is 95℃, and the recommended time is 30 sec for plasmid / virus DNA and 3 min for genomic DNA / cDNA.

b. For annealing, the recommended temperature is the Tm of the primers. If the Tm of the primers is higher than 72℃, the annealing step can be removed (two-step PCR). If necessary, annealing temperature can be further optimized in a gradient. In addition, the amplification specificity depends directly on the annealing temperature. Raising annealing temperature is helpful to improve poor amplification specificity.

c. Longer extension time is helpful to increase the amplification yield.

6.2 For Long-fragment PCR

Genie Fusion Ultra High-Fidelity DNA Polymerase can extraordinarily perform a long-fragment amplification with high specificity and yields. If the recommended program is failure to work, the following Touch Down two-step PCR may be helpful:

Steps

Temperature

Time

Cycles

Pre-denaturation

95℃

3 min

1

Denaturation

92℃

15 sec

} 5

Extension

74℃

60 sec/kb

Denaturation

95℃

15 sec

} 5

Extension

72℃

60 sec/kb

Denaturation

95℃

15 sec

} 5

Extension

70℃

60 sec/kb

Denaturation

95℃

15 sec

} 25

Extension

68℃

60 sec/kb

Final Extension

68℃

5 min

1

It is recommended to use high-quality templates and long primers . Increasing the input of template DNA may be helpful to improve the amplification yield.

For PCR Using Crude Material as Template

Genie Fusion Ultra Master Mix have a good resistance to PCR inhibitors and can be used for direct PCR amplifications of bacteria, fungi, plant tissues, animal tissues, and even whole blood samples. Crude materials that have been successfully amplified with Genie Fusion Ultra Master Mix are as follows:

Sample Type

Amplification Method

Temperature Recommendation

(for a 50 μl PCR system)

Whole Blood

Direct PCR

1 - 5 µl

Filter Paper Dry Blood

Direct PCR

1 - 2 mm2 filter paper

Cultured Cells

Direct PCR

Low amounts of cells

Yeast

Direct PCR

Single clone or 1 µl suspension

Bacteria

Direct PCR

Single clone or 1 µl suspension

Mod

Direct PCR

Low amount of sample

Sperm

Direct PCR

Low amount of sample

Plankton

Direct PCR

Low amount of sample

Plant Tissue

Direct PCR

1 - 2 mm2 tissue

Mouse Tail

PCR with lysate

1 - 5 µl lysate*

Food

PCR with lysate

1 - 5 µl lysate*

*Lysate Preparation:

Animal Tissues or Food samples

 

 

Submerge little mount of samples in lysis buffer with final concentration 200 µg/ml of Proteinase K (self-provide).

 

 

60℃ 10 min

95℃ 10 min

 

Mix well and spin at room temperature.

Collect the supernatant as lysate.

        Lysis Buffer: 20 mM of Tris-HCl, 100 mM of EDTA, 0.1% SDS, pH 8.0 (not included in this kit).

Attention:

  1. Use high-quality templates.
  2. DO NOT use dUTP or any primers or templates that contain uracil.
  3. The Phanta Max Super-Fidelity DNA Polymerase has strong proofreading activity. Therefore, the PCR products must be purified before adding A-Tailing when TA cloning.
  4. Primers design notes:

*Choose C or G as the last base of the 3’-end of the primer.

* Avoid continuous mismatching at the last 8 bases of the 3’-end of the primer.

* Avoid hairpin structure at the 3’-end of the primer.

* Tm of the primers should be within the range of 55℃ - 65℃ (recommend to calculate in Primer Premier 5), and the Tm difference between F and R primers should be less than 1℃.

* Additional sequence should not be included when calculating Tm of the primers.

* GC content of the primers should be within the range of 40% - 60%.

* The general distribution of A, G, T, C in the primers should be uniform, and avoid using regions with rich GC and rich AT.

* Keep complementary sequence less than 5 bases within the primers or between two primers, and complementary sequence less than 3 bases at the 3’-end of the primers.

* Please search the specificity of the designed primers by NCBI BLAST to avoid non-specific amplification.

Troubleshooting

No or Low Yield of PCR Products 

Primers

Optimize primer design

Annealing Temperature

Set gradient annealing temperature to find out the optimal one

Concentration of Primers

Optimize the concentration of primers

Extension Time

Optimize the extension time to 30 sec/kb-1 min/kb

Cycle Numbers

Increase cycle numbers to 35 - 40

Purity of Templates

Use high-purity templates

Template Input

Refer to the recommended reaction system and increase the input

 Unspecific or Smear Bands in Electrophoresis 

Primers

Optimize primer design

Annealing Temperature

Try to improve annealing temperature and set gradient annealing temperature to optimize

Concentration of Primers

Decrease the concentration of primers to final concentration as 0.2 µM

Extension Time

Appropriately decrease the extension time when bands longer than target bands appear

Cycle Numbers

Decrease cycle number to 25 - 30

PCR Programs

Use Two-Step PCR or Touch-down PCR

Purity of Templates

Use high purity templates

Template Input

Modify or decrease templates input referring to the recommended reaction system

Enzymes Input

Appropriately adjust or decrease the input of high-fidelity polymerase

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