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 |
100 µl |
MORV0001 |
MORV0001 |
2 X Genie Fusion Buffer |
2 X 1.25 µl |
MORV0001 |
MORV0001 |
dNTP Mix (10 mM each) |
100 µl |
MORV0001 |
MORV0001 |
10 x Loading buffer |
1.25 µl |
MORV0001 |
MORV0001 |
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