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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)
IIIIIIIV
  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 µl40.040.040.040.0
Primer 1 0.5 µM 1 µl4.04.04.04.0
Primer 2 0.5 µM 1 µl4.04.04.04.0
Template DNAb Undiluted 1 vol4 vol4 vol4 vol4 vol
25 mM 4dNTP mixc 0.2 mM 0.8 µl3.23.23.23.2
Taq polymerase 2.5 U 0.5 µl2.02.02.02.0
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)
 A B CD
  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
AdditiveOptimalVaVaVaVa
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)
EnhancerQiagenQ-Solution—proprietary
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