An efficient way to determine the presence or absence of a target sequence—often needed for genotype screening—is by visualizing end-point PCR products using agarose gel electrophoresis and ethidium bromide staining. When screening for dozens (or even hundreds) of samples, efficiency is critical, and every improvement in the workflow leads to a significant saving in time. When setting up a large number of PCR reactions, eliminating pipetting steps reduces the potential for contamination and error and minimizes repetitive strain-induced injury. Simplifying the screening procedure also enables better standardization between users, which can be crucial as screening projects scale up.

Takara Bio's PCR premixes provide the flexibility and convenience required for high sample throughput by minimizing pipetting steps. EmeraldAmp GT PCR Master Mix is suitable for amplifying GC- or AT-rich genomic DNA products up to 5 kb in length. For amplifying targets longer than 5 kb, EmeraldAmp MAX PCR Master Mix can be used, which is optimized for long targets and includes a loading dye.

EmeraldAmp GT PCR Master Mix provides excellent performance at an affordable price. PCR reactions are easy to assemble by simply adding primers and template to the 2X premix which contains a buffer, PCR polymerase, dNTPs, gel loading dyes, and a density reagent. After PCR, samples can be loaded directly onto an agarose gel for electrophoresis, used for restriction digestion, or easily cloned into a vector.

We compared the performance of Takara Bio's EmeraldAmp premixes to another dye-added premix Taq on the market, Bioline's MyTaq Red Mix. We looked at parameters such as amplification of long targets (up to 4 kb), GC-rich targets, and nonspecific amplification. The results are presented below.

Amplification of GC-rich targets

Our results showed that the non-hot-start EmeraldAmp GT and EmeraldAmp MAX PCR master mixes gave minimal background amplification with GC-rich targets. Non-hot-start MyTaq Red Mix, on the other hand, resulted in nonspecific amplification and was unable to amplify the target with a high GC content (72.3%). While EmeraldAmp GT failed to amplify the TGFB-1 gene that is 4 kb long with a 63.1% GC content, EmeraldAmp MAX was able to generate a clean and distinct band. We were unable to amplify the 4-kb TGFB-1 target using MyTaq Red Mix.

Comparison of hot-start premixes

The performance of hot-start, dye-added premixes, EmeraldAmp MAX HS and MyTaq HS Red Mix, was compared using GC-rich targets. EmeraldAmp MAX HS was able to amplify all expected products with minimal nonspecific background amplification—including long targets, regardless of GC content. In contrast, MyTaq HS Red Mix generated background amplification and failed to amplify the high GC-content gene TGFB-1, as seen in the figure below.

Four targets of various lengths and medium-to-high GC content were amplified from 50 ng of human genomic DNA with 5 different DNA polymerases using the manufacturer-specified protocol on an ABI9600 thermal cycler. See below for the PCR cycle setup for each polymerase. We performed two comparisons: one with non-hot-start enzymes (EmeraldAmp GT, EmeraldAmp MAX, and MyTaq Red Mix) and another comparison with hot-start master mixes (EmeraldAmp MAX HS and MyTaq HS Red Mix). PCR cycling conditions are shown in Table I. All PCR reactions were performed side-by-side for comparison.

Takara Bio's EmeraldAmp GT PCR Master Mix and EmeraldAmp MAX PCR Master Mix (non-hot-start and hot-start) outperformed Bioline's MyTaq Red Mix and MyTaq HS Red Mix in background amplification, target GC-content, and target length. The EmeraldAmp series gave minimal background amplification and better performance on GC-rich targets. Additionally, the EmeraldAmp MAX master mixes were able to amplify long targets while MyTaq Red Mix failed to amplify the 4-kb target gene TGFB-1.

With Takara's EmeraldAmp series of PCR master mixes, you can streamline your genotyping workflow without worrying about GC content of your target or nonspecific background amplification.