biotechrabbit™ Green Hot Start PCR Master Mix Direct Load is a perfect choice for a fast reaction setup that reduces the time required for calculation and pipetting and eliminates the need for buffer optimization. It is designed for low-background, high-throughput PCR amplification of 0.2–5 kb DNA targets. Additionally the special formulation allows reactions to be loaded directly onto gels after amplification without adding additional loading dye.

Green Hot Start PCR Master Mix contains two dyes (blue and yellow) that separate during electrophoresis, allowing migration progress to be monitored. Reactions with Green Hot Start PCR Master Mix have sufficient density for direct loading onto agarose gels.

Quality Control

Functional assay

Human genomic DNA was amplified using the Green Hot Start PCR Master Mix and specific primers to produce a distinct band.

Prevention of PCR contamination

When assembling the amplification reactions, care should be taken to eliminate the possibility of contamination with undesired DNA.

• Use separate clean areas for preparation of samples and reaction mixtures and for cycling.
• Wear fresh gloves. Use sterile tubes and pipette tips with aerosol filters for PCR setup.
• Use only water and reagents that are free of DNA and nucleases.
• With every PCR setup, perform a contamination control reaction that does not include template DNA.

Standard PCR setup

The standard PCR protocol using biotechrabbit reaction buffer provides excellent results for most applications. Optimization might be necessary for certain conditions, such as the amplification of long targets, high GC or AT content, strong template secondary structures or insufficient template purity. In such cases, optimization of template purification (see biotechrabbit nucleic acid purification kits), primer design and annealing temperature is recommended.

The best conditions for each primer-template can be optimized with the following:

• Choosing the optimal quantities of template and primers
• Optimizing cycling conditions

Basic Protocol

• The Master Mix is designed to be used without any optimization as it has all necessary reaction components in optimal amounts for successful PCR.
• Thaw on ice and mix all reagents well.
• Keep all reagents and reactions on ice.
• Pipet the master mix into thin-walled 0.2 ml PCR tubes.
• Add template and primers separately if they are not used in all reactions.

• Mix and centrifuge briefly to collect the liquid in the bottom of the tube.
• Place in the PCR cycler.

Cycling Program

• Reactions assembled with the Green Reaction Buffer have sufficient density for direct loading onto agarose gels. Do not add any loading dyes for gel loading.

Product manual

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