biotechrabbit™ lyophilized YourTaq Hot Start PCR Mix is a freeze-dried version of the well-established liquid equivalent. The stabilized format allows shipment and storage without cooling. The Master Mix is optimized for high yield of amplification of 0.1–3 kb DNA targets, even from low copy number. YourTaq Hot Start PCR Mix shows excellent PCR specificity and sensitivity for a broad range of amplicons. The mix is resistant to PCR inhibitors, such as blood (up to 20%), Ethanol or humic acid enabling PCR amplification from DNA templates with carry-over of PCR-inhibitors.

The 2× YourTaq Hot Start PCR Mix contains pure biotechrabbit YourTaq Hot Start DNA Polymerase, extremely high-quality dNTPs and optimized PCR buffer; thus, only template, PCR primers and PCR-grade water are added. In addition, the mix is suitable for amplification of GC-rich templates (up to 70%) pairing with 5× PCR Enhancer (BR1900201).

Info: Recommended annealing temperature is 2°C above primer Tm (use gradient PCR to optimize the annealing temperature).

Lyo Hot Start PCR Mix reconstitution

1) Transfer the whole content of one vial PCR Mix Reconstitution Buffer to one vial Lyo YourTaq Hot Start PCR Mix
2) Mix well – the lyophilisate will dissolve within seconds
3) Store the reconstituted YourTaq Hot Start PCR Mix at -20°C

Quality Control

Functional assay

Human genomic DNA was amplified using the 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
• Using a PCR Enhancer (i.e. BR1900201) for low amounts of template, impure or GC-rich templates
• 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.
• Optionally, use the supplied 5× PCR Enhancer to increase the yield and to lower the background in more complicated PCR reactions (low amounts of template, impure or GC-rich template).
• 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.

• For total reaction volumes other than 50 µl, scale reagents proportionally.
• Mix and centrifuge briefly to collect the liquid in the bottom of the tube.
• Place in the PCR cycler.

Cycling Program

• Add loading dye solution (see DNA Loading Dye, 6×, BR0800301) to the reactions to analyze PCR products on a gel or store them at −20°C.

Product manual

Other documentation