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Tech Note: Examples of Long-Range PCR with <i>TaKaRa LA Taq</i> DNA Polymerase

TECH NOTE

Examples of Long-Range PCR with TaKaRa LA Taq DNA Polymerase

Overview

TaKaRa LA Taq DNA Polymerase (Cat. # RR002A, also available in Hot Start and GC-Rich versions) is optimized for long-range PCR. This experiment shows examples of long-range PCR using TaKaRa LA Taq Polymerase with E. coli genomic DNA or human genomic DNA as template.

Example 1: Amplification of E. coli products 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

Peak calling and electropherograms of pre- and post-PCR size selection libraries.

Figure 1. Products 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 markers.

Example 2: Amplification of E. coli products up to 18 kb in length

Methods

PCR was performed using a TaKaRa PCR Thermal Cycler (Cat. # TP480)* with the following conditions. After PCR, 5 µl of the reaction mixture was used for electrophoresis on a 0.4% agarose gel.

Peak calling and electropherograms of pre- and post-PCR size selection libraries.

* The TaKaRa PCR Thermal Cycler is not available in all geographic locations. Please contact Technical Support for availability.

Results

Peak calling and electropherograms of pre- and post-PCR size selection libraries.

Figure 2. Products amplified using TaKaRa LA Taq from E. coli genomic DNA. 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.

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 a using TaKaRa PCR Thermal Cycler (Cat. # TP480)* with the following conditions:

Peak calling and electropherograms of pre- and post-PCR size selection libraries.

After PCR, 5 µl of the reaction mixture was used for electrophoresis on a 0.4% agarose gel.

* The TaKaRa PCR Thermal Cycler is not available in all geographic locations. Please contact Technical Support for availability.

Results

ChIP-seq library complexity and reproducibility from total DNA from small Amounts of cells. DNA SMART ChIP-seq library overlap with ENCODE data.

Figure 3. Products amplified by TaKaRa LA Taq from human genomic DNA. 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 products up to 38 kb in length or human products up to 27 kb in length was achieved using TaKaRa LA Taq DNA Polymerase.

Learn more about TaKaRa LA Taq DNA Polymerase>>

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