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Alongside the essential ingredients of PCR - template, primers, polymerase, and dNTPs - a variety of additives has been identified that show strong effects upon yield, specificity and consistency of PCR (Table 1). Each of these may have a beneficial effect on individual amplifications but it is still largely impossible to predict their effects a priori. Therefore, an individual assay optimization may be essential for cumbersome template-primer sets.
In order to avoid such tedious optimizations we developed High Yield Buffer that contains a balanced composition of PCR enhancers (please see our PCR Additives Kit) for maximum efficiency. High Yield Buffer
- decreases the melting temperature of dsDNA and primer/ssDNA complexes and thus, achieves superior amplification results especially for "difficult" (GC-rich) template-primer combinations (dNTP Mix GCamplifier),
- facilitates incorporation of labeled/modified nucleotides into the newly synthesized DNA strand,
and is therefore recommended for applications that require high yields (Fig. 1) and for PCR-mediated DNA labeling.
| Taq Pol
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Taq Pol / high yield buffer
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200 bp
DNA Ladder
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| 100 pg
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50 pg
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20 pg
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10 pg
|
100 pg
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50 pg
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20 pg
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10 pg
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Figure 1: High yield buffer yields 3-10fold more PCR product compared to conventional buffer but the increased yield may come at the costs of reduced specificity. Assay: Amplification of lambda DNA (template dilution series), 1 kb fragment, 1.25 units Taq Pol / reaction, 95°C, 2 min; 30x (95°C, 10 sec; 59°C, 20 sec; 72°C, 1 min); 72°C, 1 min
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For those PCRs in which High Yield Buffer still does not yield satisfactory results we offer the most prominent PCR enhancers (Table 1) in our PCR Additives Kit for individual optimization. They affect stability of polymerases, mediate polymerase/template contact or reduce secondary structures of template or primers for improved exposure. Although widely reported in the literature, their benefits on a particular PCR reaction must in most cases be determined empirically.
| Enhancer
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Recommended concentration
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| DMSO
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2 - 10%
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| Betaine
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1 - 1.5 M
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| Formamide
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1 - 5%
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Non-ionic detergents
(e.g. Triton X-100, Tween 20 or Nonidet P-40)
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0.1 - 1%
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| TMAC (tetramethylammonium chloride)
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15 - 100 mM
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| 7-deaza-2'-deoxyguanosine(dC7GTP)
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3 : 1 (dC7GTP : dGTP)
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| BSA (bovine serum albumin)
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0.1 - 0.5 µg/µl
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| SSB (single-stranded DNA binding protein)
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0.2 - 5 ng/µl
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| Pyrophosphatase
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0.001 - 0.1 U/reaction
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Table 1: Most prominent PCR enhancers
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LAROVA GmbH