High Fidelity DNA Polymerase Protocol

High Fidelity DNA Polymerase Protocol

Genie Fusion Ultra High-Fidelity DNA Polymerase

Package Information

Components MORV0001 100 U MORV0002 500 U MORV0003 1000 U

Genie Fusion Ultra High-Fidelity DNA Polymerase
(1 U/µl)

100 µl

MORV0001
x 5

MORV0001
x 5

2 X Genie Fusion Buffer

2 X 1.25 µl

MORV0001
x 5

MORV0001
x 5

dNTP Mix (10 mM each)

100 µl

MORV0001
x 5

MORV0001
x 5

10 x Loading buffer

1.25 µl

MORV0001
x 5

MORV0001
x 5

1. Experimental Process

1.1 Standard PCR

Recommended PCR System:
Keep all components on ice during the experiment. All components need to be mixed thoroughly after thawing and put back at -20℃ immediately after using.

Components Amounts

ddH2O

up to 50 μl

2× Genie Fusion Buffer a

25 μl

dNTP Mix (10 mM each)

1 μl

Primer 1 (10μM)

2 μl

Primer 2 (10μM)

2 μl

Genie Fusion Ultra High-Fidelity DNA Polymerase (1 U/μl)

1 μl

Template DNA b

X μl

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

25 - 35

Extension c

72 ℃

30 - 60 sec/kb

25 - 35

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 Long Range PCR

Genie Fusion Ultra High-Fidelity DNA Polymerase can perform a long-range amplification with high specificity and yields. If the standard PCR protocol (Section 6.1) yields insufficient results, the following Touch-Down, two-step PCR is recommended.

Steps Temperature Time Cycles

Pre-denaturation a

95℃

3 min

1

Denaturation

92℃

15 sec

5

Extension

74℃

60 sec/kb

5

Denaturation

95℃

15 sec

5

Extension

72℃

60 sec/kb

5

Denaturation

95℃

15 sec

5

Extension

70℃

60 sec/kb

5

Denaturation

95℃

15 sec

25

Extension

68℃

60 sec/kb

25

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.

6.3 Direct PCR with Crude Samples

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

Sample Type Amplification Method Temperature Recommendation (for a 50 μl PCR system)1 - 5 µl lysate*

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

Extension

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