Takara LA Taq DNA polymerase (Cat. # RR002A, also available in hot-start and high GC-compatible versions) is optimized for long-range PCR. These experiments show examples of long-range PCR using Takara LA Taq Polymerase with E. coli or human genomic DNA as the template.
Tech Note
Long-range PCR with Takara LA Taq DNA polymerase
Introduction
38 kb E. coli DNA
Example 1: Amplification of E. coli DNA up to 38 kb in length
Methods
100 ng of E. coli genomic DNA was used as the template in a 50-µl reaction. Takara LA Taq DNA polymerase (Cat. # RR002A) was used for amplification using the recommended conditions. After PCR, 5 µl of the reaction mixture was used for electrophoresis on a 0.4% agarose gel.
Results
Figure 1. Products (20–38 kb) amplified from E. coli genomic DNA using Takara LA Taq DNA polymerase. Amplified product sizes are 20 kb (Lane 2), 28 kb (Lane 3), 30 kb (Lane 4), and 38 kb (Lane 5). Molecular weight markers: Lane 1: HindIII-digested lambda DNA; Lane 6: High-molecular weight marker.
18 kb E. coli DNA
Example 2: Amplification of E. coli DNA up to 18 kb in length
Methods
PCR was performed using the following conditions outlined below. After PCR, 5 µl of the reaction mixture was used for electrophoresis on a 0.4% agarose gel.
Results
Figure 2. Products (2–18 kb) amplified from E. coli genomic DNA using Takara LA Taq. Amplified product sizes are 2 kb (Lane 2), 4 kb (Lane 3), 6 kb (Lane 4), 8 kb (Lane 5), 10 kb (Lane 6), and 18 kb (Lane 7). Lanes 1 and 8 contain HindIII-digested lambda DNA as a molecular weight marker.
Human gDNA
Example 3: Long-range PCR using human genomic DNA
Methods
The TPA gene region and the β-globin cluster region were each amplified from human genomic DNA using Takara LA Taq DNA polymerase. The reactions were set up as indicated below:
| Component | Volume | Final concentration |
|---|---|---|
| Human genomic DNA (500 ng/µl) | 1 µl | 500 ng per 50 µl reaction |
| 10x LA PCR Buffer II (Mg2+ Plus) | 5 µl | 1X |
| dNTPs | 8 µl | 400 µM each |
| Sense Primer (20 pmol/µl) | 0.5 µl | 0.2 µM |
| Antisense Primer (20 pmol/µl) | 0.5 µl | 0.2 µM |
| Takara LA Taq | 0.5 µl | 2.5 U per 50 µl reaction |
| Sterile distilled water | 34.5 µl |
After the 50-µl reactions were prepared, each reaction was overlayed with an equal volume of mineral oil. PCR was performed using the following conditions:
After PCR, 5 µl of the reaction mixture was used for electrophoresis on a 0.4% agarose gel.
Results
Figure 3. Products amplified from human genomic DNA using Takara LA Taq. LA Taq was used to amplify 17.5-kb and 21.5-kb fragments of the beta-globin gene cluster region (Lanes 2 and 3, respectively) and a 27-kb portion of the TPA gene region (Lane 4). Molecular weight markers: Lane 1: HindIII-digested lambda DNA; Lane 5: High-molecular weight markers.
Conclusion
Amplification of E. coli genomic targets up to 38 kb in length or human genomic targets up to 27 kb in length was achieved using Takara LA Taq DNA polymerase.
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