PCR Buffer
An optimal buffer system is essential to perform successful PCR. Reliable PCR results depend on many factors: the quality of the DNA and primers as well as the PCR instrument itself.
Ampliqon has developed different Tris-based buffer solutions to meet different requirements in PCR applications. The buffers are commonly supplied in 10x formulations with 15 mM MgCl2 included. Ampliqon buffers are also available without Mg2+ and Tween 20 or Triton X-100.
Buffer convenience and user-flexibility is achieved by the ability to choose either the right DNA Polymerase/buffer combination or the optimal buffer based Master Mix among the wide range of Ampliqon’s DNA polymerase products. All Ampliqon DNA Polymerases and Master Mixes are available with different buffer options, making it easy to choose either the right combination of DNA polymerase and buffer or the correct Master Mix, for matching different PCR applications and PCR assay conditions.
- Ammonium Buffer
- Standard Buffer
- Combination Buffer
- 5x PCR Buffer RED
- 4x GC Buffer I & 4x GC Buffer II
- PCR Grade Water
- Betaine Enhancer Solution 5 M
Ampliqon recommends Ammonium Buffer for most PCR applications. It promotes robust amplification, high yield and high specificity.
Ammonium Buffer
Ammonium Buffer is recommended for most PCR applications. It results in high yield of PCR products and minimises the need for optimisation of Mg2+ concentrations or the annealing temperatures.
FEATURES
- Promotes amplification on most DNA templates
- High yield
- High specificity
- Tolerance for primer annealing temperature
- 10x Buffer
AVAILABLE IN 4 FORMULATIONS:
- 15 mM MgCl2,
- Mg2+ free
- Tween free
- Mg2+ + Tween free
DESCRIPTION
Ammonium Buffer promotes high specificity over a broad range of annealing temperatures and Mg2+ concentrations. Ammonium Buffer also works well when dealing with difficult templates, e.g. GC-rich DNA sequences.
TIP - CHOOSE THE RIGHT BUFFER
Mg2+ free buffer is recommended if you need to optimise Mg2+ concentrations in your PCR set-up, especially if your application requires Mg2+ concentration lower than 1.5 mM.
Detergent free buffers are recommended for automation and downstream applications involving fluorescent spectrometry.
PERFORMANCE OF AMPLIQON PCR BUFFER SYSTEM
Ammonium Buffer: Minimal need for optimisation.
A broad range of Mg2+ concentrations and temperatures results in specific products with high yield (Lanes 1.5 – 2.5 and lanes 57-66)
Standard Buffer: Optimisation is needed.
A narrow range of Mg2+ concentrations and temperatures results in specific products. (Lanes 1.5 – 2.0 and lanes 60-66)
Combination Buffer: Optimisation is needed.
A narrow range of Mg2+ concentrations and temperatures results in specific products with high yields. (Lane 1.5 and lanes 60-66)
Examples of PCR amplification of ENG9. TEMPase and the indicated buffers were used at the indicated Mg2+ concentrations or temperatures. The first part shows a Mg2+ dilution series from 0.5 – 4.5 mM MgCl2, at 60°C. The second part shows a temperature gradient from 51 – 66 °C at 1.5 mM MgCl2. M: marker.
Standard Buffer
Standard Buffer is the traditional potassium buffer and promotes high specificity.
FEATURES
- Promotes reliable amplification on most DNA templates
- High specificity
- 10x Buffer
AVAILABLE IN 4 FORMULATIONS:
- 15 mM MgCl2,
- Mg2+ free
- Triton-X 100 free
- Mg2++ Triton-X 100 free
DESCRIPTION
Ammonium Buffer is recommended for most PCR applications however; we recommend that you continue using Standard Buffer if you have already optimised your protocols for this buffer. The use of Standard buffer most often requires optimisation of primer annealing temperatures and Mg2+ concentrations. Highly pure DNA templates are preferable if you use this buffer.
TIP - CHOOSE THE RIGHT BUFFER
Ammonium Buffer is recommended for most PCR applications. It results in high yield of PCR products and minimises the need for optimisation of Mg2+ concentrations or the annealing temperatures.
Mg2+ free buffer is recommended if you need to optimise Mg2+ concentrations in your PCR set-up, especially if your application requires Mg2+ concentration lower than 1.5 mM.
Detergent free buffers are recommended for automation and downstream applications involving fluorescent spectrometry.
PERFORMANCE OF AMPLIQON PCR BUFFER SYSTEM
PERFORMANCE OF AMPLIQON PCR BUFFER SYSTEM
Ammonium Buffer: Minimal need for optimisation.
A broad range of Mg2+ concentrations and temperatures results in specific products with high yield (Lanes 1.5 – 2.5 and lanes 57-66)
Standard Buffer: Optimisation is needed.
A narrow range of Mg2+ concentrations and temperatures results in specific products. (Lanes 1.5 – 2.0 and lanes 60-66)
Combination Buffer: Optimisation is needed.
A narrow range of Mg2+ concentrations and temperatures results in specific products with high yields. (Lane 1.5 and lanes 60-66)
Examples of PCR amplification of ENG9. TEMPase and the indicated buffers were used at the indicated Mg2+ concentrations or temperatures. The first part shows a Mg2+ dilution series from 0.5 – 4.5 mM MgCl2, at 60°C. The second part shows a temperature gradient from 51 – 66 °C at 1.5 mM MgCl2. M: marker.
Combination Buffer
Combination Buffer is a balanced ammonium-potassium PCR buffer. Combination Buffer results in tolerance towards optimization of primer annealing temperatures and Mg2+ concentrations.
FEATURES
- Promotes reliable amplification on most DNA templates
- Tolerance toward primer annealing temperature
- 10x Buffer
AVAILABLE IN 4 FORMULATIONS:
- 15 mM MgCl2,
- Mg2+ free
- Tween free
- Mg2+ + Tween free
DESCRIPTION
Ammonium Buffer is recommended for most PCR applications however; we recommend you to use Combination Buffer as it show good results on certain PCR instruments. Combination Buffer is also worth testing when searching for PCR buffers to be used in new set-up.
TIP - CHOOSE THE RIGHT BUFFER
Ammonium Buffer is recommended for most PCR applications. It results in high yield of PCR products and minimises the need for optimisation of Mg2+ concentrations or the annealing temperatures.
Mg2+ free buffer is recommended if you need to optimise Mg2+ concentrations in your PCR set-up, especially if your application requires Mg2+ concentration lower than 1.5 mM.
Detergent free buffers are recommended for automation and downstream applications involving fluorescent spectrometry.
PERFORMANCE OF AMPLIQON PCR BUFFER SYSTEM
PERFORMANCE OF AMPLIQON PCR BUFFER SYSTEM
Ammonium Buffer: Minimal need for optimisation.
A broad range of Mg2+ concentrations and temperatures results in specific products with high yield (Lanes 1.5 – 2.5 and lanes 57-66)
Standard Buffer: Optimisation is needed.
A narrow range of Mg2+ concentrations and temperatures results in specific products. (Lanes 1.5 – 2.0 and lanes 60-66)
Combination Buffer: Optimisation is needed.
A narrow range of Mg2+ concentrations and temperatures results in specific products with high yields. (Lane 1.5 and lanes 60-66)
Examples of PCR amplification of ENG9. TEMPase and the indicated buffers were used at the indicated Mg2+ concentrations or temperatures. The first part shows a Mg2+ dilution series from 0.5 – 4.5 mM MgCl2, at 60°C. The second part shows a temperature gradient from 51 – 66 °C at 1.5 mM MgCl2. M: marker.
5x PCR Buffer RED
5x PCR Buffer RED is a ready to use PCR buffer, including all components for standard PCR applications.
FEATURES
- All in one Buffer for highest convenience
- Direct gel loading onto agarose gels
- Red colour enables easy visualisation of pipetting and loading
- Dye front runs at 1000 – 2000 bp on DNA 0.5 – 1.5 % agarose
- Same specifications as Ammonium Buffer
- 5x Buffer
DESCRIPTION
5x PCR Buffer RED offers the same specification as Ammonium Buffer. It results in high yield of PCR products and minimises the need for optimisation of Mg2+ concentrations or the annealing temperatures. The included red dye and density reagent, eliminates the need for loading dye as well as the time consuming sample preparation before electrophoresis.
DIRECT GEL LOADING
5X PCR BUFFER RED PROMOTES SPECIFIC AND CLEAR RESULTS
5x PCR Buffer RED promotes specific and clear results. The same DNA target was evaluated using the same PCR set-up in duplicates amplified by either Ampliqon Taq DNA Polymerase or TEMPase Hot Start DNA Polymerase.
4x GC Buffer I & 4x GC Buffer II
GC Buffer I and GC Buffer II is developed to promote excellent amplification in combination with TEMPase Hot Start DNA Polymerase of targets with varying degrees of GC content.
FEATURES
- Promotes amplification of GC-rich DNA
- 4x PCR buffer
- Promotes diminished formation of non-specific PCR products
DESCRIPTION
Ammonium Buffer is often sufficient to promote amplification on most DNA targets. If Ammonium Buffer fails to promote amplification on GC-rich or difficult DNA templates we recommend to change to GC Buffer I in combination with TEMPase Hot Start DNA Polymerase. If your amplification is still not acceptable, then switch to GC Buffer II.
TIP - CHOOSE THE RIGHT BUFFER
Ammonium Buffer is recommended for most PCR applications. It results in high yield of PCR products and minimises the need for optimisation of Mg2+ concentrations or the annealing temperatures.
Mg2+ free buffer is recommended if you need to optimise Mg2+ concentrations in your PCR set-up, especially if your application requires Mg2+ concentration lower than 1.5 mM.
Detergent free buffers are recommended for automation and downstream applications involving fluorescent spectrometry.
OPTIMISATION OF GC-RICH DNA AMPLIFICATION
PCR Grade Water
Ampliqon offers ultrapure PCR grade water.
FEATURES
- Ultrapure H2O
- Free of endonuclease, nicking and exonuclease activity
- Free of human DNA
DESCRIPTION
The PCR grade water is ultrapure H2O, free of endonuclease-, nicking-, and exonuclease activity, free of human DNA.
Tested for contaminating activities, with no trace of endonuclease activity, nicking activity or exonuclease activity. Absence of human DNA is tested by PCR.
Betaine Enhancer Solution 5 M
Betaine Enhancer Solution is one of the most effective additives over a wide range of different templates, including GC-rich sequences and templates known to be extremely difficult to amplify. Betaine enhancer solution lowers the DNA melting temperature and has an enhancing effect on the polymerase.
FEATURES
- Enhances amplification on GC-rich and difficult DNA targets
- Lowers the melting temperature
DESCRIPTION
The performance of Betaine Enhancer Solution is superior compared to standard enhancers such as formamide, DMSO, TMAC, BSA and non-ionic detergents. Betaine Enhancer Solution is an excellent enhancer especially when used with GC-rich regions or templates with a high degree of secondary structures.
In detail, Betaine binds preferentially to AT rich sequences in the major groove, thereby stabilizing AT rich regions of the DNA. Because AT forms 2 hydrogen bonds and GC forms 3, the bonding of AT is less stable than the one of GC. As a consequence of the stabilizing effect of Betaine on AT bonding, the stability of AT bonding and GC bonding is brought close to an equal level. At the same time, Betaine has a sequence independent destabilizing effect on all DNA. Summarized, the Tm of AT rich and GC rich sequences are equalized and the overall Tm is lowered. Furthermore, Betaine aids the processivity of thermostable polymerases and reduces “pauses” in polymerization caused by secondary structure that can induce the polymerase to disassociate from the DNA strand.
Betaine has a decreasing effect on the melting temperature of DNA and primers. Therefore, denaturation temperatures as well as primer annealing temperatures should be reduced by 1 – 5 °C. The optimal annealing temperature should be determined individually for each reaction.
