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UNIT 15.1 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization

  1. Martha F. Kramer,
  2. Donald M. Coen

Published Online: 1 NOV 2001

DOI: 10.1002/0471142727.mb1501s56

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Kramer, M. F. and Coen, D. M. 2001. Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Molecular Biology. 56:15.1:15.1.1–15.1.14.

Author Information

  1. Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 NOV 2001
  2. Published Print: OCT 2001
Table 15.1.1. Master Mixes for Optimizing Reaction Components
ComponentsFinal concentrationPer reactionMaster mixa (µl)
  1. a

    Total volume = 360 µl (i.e., enough for n + 1 reactions).

  2. b

    Template DNA volume (“vol”) is generally 1 to 10 µl.

  3. c

    If 2 mM 4dNTP mix is preferred, use 10 µl per reaction, or 40 µl for each master mix; adjust the volume of water accordingly.

  4. d

    Substitute with other enhancer agents (see recipe in Reagents and Solutions) as available.

10× MgCl2-free PCR buffer 1× 10 µl40.
Primer 1 0.5 µM 1 µl4.
Primer 2 0.5 µM 1 µl4.
Template DNAb Undiluted 1 vol4 vol4 vol4 vol4 vol
25 mM 4dNTP mixc 0.2 mM 0.8 µl3.
Taq polymerase 2.5 U 0.5 µl2.
DMSOd (20×) 5% 5 µl —20.0 — —
Glycerold (10×) 10% 10 µl — —40.0 —
PMPEd (100×) 1% 1 µl — — —4.0
H2O — To 90 µlTo 360To 360To 360To 360
Table 15.1.2. Master Mixes for Optimizing First-Cycle Reactions
ComponentsFinal concentrationMaster mix (µl)
  1. a

    V, variable amount (total volume should be 100 µl).

  2. b

    Use undiluted or diluted template DNA based on results obtained in step 6.

10× PCR buffer10101010
MgCl2 (L, M, or H)Optimal10101010
Primer 10.5 µM 1.0 1.0 1.0 1.0
Primer 20.5 µM 1.0 1.0 1.0 1.0
Template DNA — bVaVaVaVa
25 mM 4dNTP mixb0.2 mM 0.8 0.8 0.8 0.8
Taq polymerase2.5 U 0.5 0.5
Taq pol + TaqStart2.5 U — 1.0
H2OTo 100 µlVaVaVaVa
Preparation temperature Room temperatureIce slurryRoom temperatureRoom temperature
Table 15.1.3. PCR Optimization Products
Optimization goalSupplierProduct
Optimization supportPerkin-ElmerTechnical information in appendix to catalog
Optimization supportPromegaPCR troubleshooting program on the Internet: http://www.promega.com/amplification/assistant
Optimization kitsBoehringer-Mannheim, Invitrogen, Stratagene, Sigma, Epicentre Technologies, Life TechnologiesSeveral buffers, Mg2+, and enhancers which may include DMSO, glycerol, formamide, (NH4)2SO4, and other unspecified or proprietary agents
Quick startupAmersham Pharmacia BiotechReady-To-Go Beads “optimized for standard PCR” and Ready-To-Go RAPD Analysis Beads (buffer, nucleotides, Taq DNA polymerase)
Quick startupFisherEasyStart PCR Mix-in-a-Tube—tubes prepackaged with wax beads containing buffer, MgCl2, nucleotides, Taq DNA polymerase
Quick startupLife TechnologiesPCR SuperMix—1.1× conc.—premix containing buffer, MgCl2, nucleotides, TaqDNA polymerase
Quick startupMarsh BiomedicalAdvanced Biochemicals Red Hot DNA Polymerase—a new rival for Taq polymerase with convenience features
Hot-start/physical barrierFisher, Life TechnologiesMolecular Bio-Products HotStart Storage and Reaction Tubes—preadhered wax bead in each tube; requires manual addition of one component at high temperature
Hot-start/separate MgCl2InvitrogenHotWax Mg2+ beads—wax beads contain preformulated MgCl2 which is released at first elevated-temperature step
Hot-start/separate MgCl2StratageneStrataSphere Magnesium Wax Beads—wax beads containing preformulated Mg2+
Hot Start/separate polymerasePromegaTaqBead Hot Start Polymerase—wax beads encapsulating Taq DNA polymerase which is released at first elevated-temperature step
Hot-start/reversible inactivation of polymerase by antibody bindingClontechTaqStart Antibody, TthStart Antibody—reversibly inactivate Taq and Tth DNA polymerases until first denaturation at 95°C
Hot-start/antibody bindingLife TechnologiesPlatinumTaq—contains PlatinumTaq antibody
Hot-start/antibody bindingSigmaJumpStart Taq—contains TaqStart antibody
Hot-start/reversible chemical modificationPerkin-ElmerAmpliTaq Gold—activated at high temperature
Hot-start/reversible chemical modificationQiagenHotStarTaq DNA Polymerase—activated at high temperature
EnhancerBoehringer Mannheim, New England BiolabsTth pyrophosphatase, thermostable
EnhancerClontechGC-Melt (in Advantage-GC Kits)—proprietary
EnhancerCPGTaq-FORCE Amplification System and MIGHTY Buffer—proprietary
EnhancerFisherEppendorf MasterTaq Kit with TaqMaster Enhancer—proprietary
EnhancerLife TechnologiesPCRx Enhancer System—proprietary
EnhancerPromegaE.coli Single Stranded Binding Protein (SSB)
EnhancerStratagenePerfect Match Polymerase Enhancer—proprietary
EnhancerStratageneTaqExtender PCR Additive—proprietary
Table 15.1.4. Thermostable DNA Polymerases
DNA polymeraseBiological sourceSupplierProduct endsExonuclease activity
Generic nameTrade name
  1. a

    No information at this time.

PfuPyrococcus furiosusStratagene, PromegaBlunt3′-5′ (proofreading)
Pfu (exo-)Pyrococcus furiosusStratageneBluntNo
PspDeep VentPyrococcus sp.GB-DNew England BiolabsBlunt3′-5′ (proofreading)
Psp (exo-)Deep Vent (exo-)Pyrococcus sp.GB-DNew England BiolabsBluntNo
Pwo Pyrococcus woeseiBoehringer MannheimBlunt3′-5′ (proofreading)
Taq (native and/or recombinant)Thermus aquaticusAmbion, Amersham Pharmacia Biotech, Boehringer Mannheim, Clontech, Fisher, Life Technologies, Marsh Biomedical, Perkin Elmer, Promega, Qiagen, Sigma, Stratagene3′A5′-3′
Taq, N-terminal deletionStoffel fragment Klen-TaqThermus aquaticusPerkin-Elmer, Sigma3′ANo
TbrDyNAzymeThermus brocianusMJ Researcha5′-3′
Tfl Thermus flavusPromega, Epicentre TechnologiesBlunta
TliVentThermococcus litoralisNew England Biolabs (Vent), PromegaBlunt3′-5′ (proofreading)
Tli (exo-)Vent (exo-)Thermococcus litoralisNew England BiolabsBluntNo
TmaUlTmaThermotoga maritimaPerkin-ElmerBlunt3′-5′ (proofreading)
TthThermus thermophilusAmersham Pharmacia Biotech, Boehringer Mannheim, Epicentre Technologies, Perkin Elmer, Promega3′ A5′-3′
Table 15.1.5. Thermostable DNA Polymerase Blends
Product (trade name)SupplierThermostable DNA polymerases and other components
Expand High Fidelity, Expand Long Template, and Expand 20kb PCR SystemsBoehringer MannheimTaq + Pwo
KlenTaq LA Polymerase MixClontech, SigmaKlenTaq-1 (5′-exonuclease deficient Taq) + unspecified proofreading polymerase
Advantage-HF PCR KitClontechKlenTaq-1 + unspecified proofreading polymerase + TaqStart Antibody
Advantage-cDNA and Advantage-GC cDNA Polymerase Mixes and KitsClontechKlenTaq-1 + unspecified proofreading polymerase + TaqStart Antibody; GC Kit contains GC Melt
Advantage Genomic and Advantage-GC Genomic Polymerase Mixes and KitsClontechTth + unspecified proofreading polymerase + TthStart Antibody; GC Kit contains GC Melt
eLONGase Enzyme MixLife TechnologiesTaq +Psp + unspecified proofreading polymerase(s) + eLONGase Buffer
Platinum Taq DNA PolymeraseLife TechnologiesTaq + Psp + Platinum Taq Antibody
Platinum High Fidelity DNA PolymeraseLife TechnologiesTaq + Psp + Taq Antibody
DyNAzyme EXT PolymeraseMJ ResearchTbr with unspecified enhancer
GeneAmp XL PCR and XL RNA PCR KitsPerkin-ElmerTth + Tli
OmniBase Sequencing Enzyme MixPromegaUnspecified proofreading polymerase(s) with thermostable pyrophosphatase
AccuTaq LA DNA Polymerase MixSigmaTaq + unspecified proofreading polymerase
TaqPlus Long and TaqPlus Precision PCR SystemsStratagenePfu+Taq; TaqPlus Precision Reaction Buffer (proprietary)
Accurase Fidelity PCR Enzyme Mix Calypso High Fidelity Single Tube RT-PCR SystemTetralinkThermus sp.; + Thermococcus sp. Calypso also contains AMV-RT