Polymerase Chain Reaction (PCR) simply involves the selective amplification of a region of DNA. Two short oligonucleotide primers are annealed to the DNA molecule, and these delimit the region to be amplified. Amplification is carried out by a DNA polymerase enzyme which synthesises new complimentary strands of DNA.
PCR can often prove problematic particularly when attempting to amplify long DNA regions or DNA templates with difficult nucleotide composition. Some DNA polymerases are unable to overcome such difficulties and may cause premature termination of complementary strand synthesis by misincorporations or pyrophosphorolysis of phosphodiester bonds in the template DNA. Moreover, some polymerases may become inactivated by the accumulation of pyrophosphate in the reaction mixture and the increased thermal stress (due to longer reaction times in the extension step).
One method to successfully amplify ‘difficult’ DNA template, when using any DNA polymerase, is to incorporate a special additive into the reaction mixture. VH Bio’s Band Doctor is one such additive which enhances polymerase performance and DNA amplification and is particularly suitable for ‘difficult’ templates with high GC content and complex structures. The benefits of using Band Doctor for DNA amplification include:
-
optimised activity, specificity and sensitivity
-
higher fidelity, higher yield and higher thermal stability of template replication
-
correction of nucleotide misincorporation
-
prevention of undesirable pyrophosphate build up
-
enhanced synthesis of very long DNA regions (>10,000bp) even from trace amounts of template DNA
-
provides optimal pH leading to an improved yield / background ratio
-
substantial reduction in non-specific background (gel smears), false bands and spurious results
-
particularly appropriate when amplifying using a DNA polymerase exhibiting inherent 3’-5’ proof-reading activity
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suitable for applications where accurate replication of the template sequence is essential, such as sequencing, direct cloning and expression
-
ideal for Next Generation Sequencing studies e.g. generating amplicons from GC rich genes
Band Doctor is supplied as a 5X concentrated solution and is recommended for use in the reaction mixture at a final concentration of 0.5X – 2X.
Reaction Mixture (concentration of Band Doctor):
|
Component |
Volume (in µl) |
BD 0.5X
Final Conc. |
BD 1X
Final Conc.
|
BD 1.5X
Final Conc. |
BD 2X
Final Conc. |
|
10X Polymerase Reaction Buffer |
5 |
5 |
5 |
5 |
|
10mM dNTP Mastermix |
1 |
1 |
1 |
1 |
|
Forward Primer (10pmol/µl)
|
2 |
2 |
2 |
2 |
|
Reverse Primer (10pmol/µl) |
2 |
2 |
2 |
2 |
|
Template DNA |
x |
x |
x |
x |
|
5X Band Doctor |
5 |
10 |
15 |
20 |
|
DNA Polymerase (2.5U/µl) |
0.5 |
0.5 |
0.5 |
0.5 |
|
Sterile, distilled water |
up to 50 |
up to 50 |
up to 50 |
up to 50 |
Price
BD50 Band Doctor 50 ml
£ 600.00 (excluding VAT and packaging)
50 ml Band Doctor will typically provide 2,500 reactions (50 µl volume) at 2X concentration.
Smaller aliquots are available on request. For pricing please enquire.
Click here for information on VH Bio’s range of DNA polymerases suitable for Real Time and standard PCR.
For further information please contact our Molecular Biology Product Manager, Catherine Mair (email: cmair@vhbio.com or tel: +44 (0)191 495 8211).
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