Q7.  WHAT IS THE OPTIMAL CONCENTRATION OF ENZYME FOR LA PCR?

In general, 2.5 units of either TaKaRa LA Taq or TaKaRa Ex Taq are used in a 50 reaction. The optimum amount of enzyme may be adjusted depending on the amount or the degree of complexity of template DNA, or the DNA lengths to be amplified.

In the case of excess enzyme, non-specific reactions may occur and/or visualization of electrophoresis may be hampered which results in a diffuse smear. The efficiency of amplification may be diminished when the enzyme concentration in the reaction is low.

The following results show how the amount of enzyme affects LA PCR amplifications.

1. Amplification of DNA with TaKaRa LA Taq

Template:
DNA
2.5 ng/50 PCR

Amplified sizes:
20 kbp, 28 kbp, 30 kbp

Applied volume:
6

1% Agarose gel

electrophoresis 

2. Amplification of human genomic DNA with TaKaRa LA Taq

 Template:
Human genomic DNA

500 ng/50 PCR

Amplified region:
-globin cluster

Amplified sizes:
17.5 kbp, 21.5 kbp , 22.5 kbp

Applied volume:
6

1% Agarose gel

electrophoresis