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Long Range PCR Master Mix, 2×

Features

  • Optimized Long Range PCR Master Mix for minimal hands-on and fast setup
  • Mix of pure polymerases and highest quality dNTPs for high yield and short cycle times
  • Increased fidelity for accurate amplification of GC-rich templates

Applications

  • High-throughput, long-range PCR up to 40 kb
  • Amplification of GC-rich templates

Purchase Long Range PCR Master Mix, 2×

Availability: In stock Bulk requests please inquire at oem@biotechrabbit.com

Catalog # Size Package Content Price* Qty
BR0300401 100 rxn of 50 µl 2 × 1.25 ml Long Range PCR Master Mix
1.5 ml 5× PCR Enhancer
€107.00
BR0300402 500 rxn of 50 µl 10 × 1.25 ml Long Range PCR Master Mix
1.5 ml 5× PCR Enhancer
€450.00

*Does not include applicable tax, shipping & handling, or other charges.

OR

biotechrabbit™ Long Range PCR Master Mix is a perfect choice for fast reaction setup for long-range PCR that reduces the time required for calculation and pipetting and eliminates the need for buffer optimization. It is designed for amplification of targets up to 40 kb in size. The master mix works well with GC-rich templates and amplifies DNA with a higher fidelity than Taq DNA polymerase.

The Long Range PCR Master Mix contains a blend of thermophilic polymerases, extremely high-quality dNTPs and optimized PCR buffer; thus, only template, PCR primers and PCR-grade water are added.

For the most demanding applications, the supplied 5× PCR Enhancer can be optionally used to improve results when using templates with GC-rich sequences and complex structures.

Long Range PCR Master Mix produces a mixture of A-tailed and blunt-end PCR products. It is advisable to blunt products before cloning into the blunt-end vector.

 

Component

Composition

Long Range PCR Master Mix

Optimized 2× Long Range PCR Master Mix

5× PCR Enhancer

Proprietary PCR enhancer mix

 

STORAGE

−20°C (until expiry date – see product label)

 

Quality Control
Functional assay

Human genomic DNA was amplified using the Long Range 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.

Component

Volume

Final concentration

Long Range PCR Master Mix (2×)

25 µl

5× PCR Enhancer (optional - BR1900201)

10 µl

Forward primer

Variable

0.2–1 µM

Reverse primer

Variable

0.2–1 µM

Template DNA

Variable

10 pg–1 μg

 

Use 0.01–1 ng for plasmid or phage DNA and 0.1–1 μg for genomic DNA

Nuclease free water

Variable

 

Total volume

50 µl

 

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

Step

Temperature

Time

Cycles

Initial activation

95°C

2 min

1

Denaturation

95°C

30 s

25–35

Annealing

55°C

30–45 s

25–35

 

Approximately 5°C below Tm of primers

Extension

72°C

30 s/kb

25–35

For fragments longer than 5 kb, use 68°C extension temperature and 1 min/kb timing.

Final extension

72°C

5 min

1

 

To extend all incomplete PCR products

Storage in the cycler

4°C

Indefinitely

1

  • Add loading dye solution (see DNA Loading Dye, 6×, cat. no. BR0800301) to the reactions to analyze PCR products on a gel or store them at −20°C.
  • For cloning, always purify the PCR product from a gel (see BR0700401 GenUP™ Gel Extraction Kit).
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