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PCR Basics
PCR Basics
1. 2. 3. 4.
5.
Experimental Notes
Analyze single DNA fragments out of large complex mixture. [ Human genome mixture of 12 million 300bp fragments]
Alter DNA sequence directed mutagenesis.
Overview of PCR
1. Temperature Cycling Denaturation Annealing Extension 94 55 72
PCR Amplification
Exponential Amplification
dNTP
(dATP, dTTP, dCTP, dGTP)
PCR Variables
1. 2. 3. 4. 5. Temperature Cycle Times and Temps Primer Buffer Polymerase
Temperature
Denaturation
Trade off between denaturing DNA and not denaturing Taq Polymerase Taq half-life 40min at 95 , 10min at 97.5 95
Annealing
Trade off between efficient annealling and specificity 2-5 below Tm
Extension
Temperature optimum for Taq Polymerase 72
1 2 3 4 5 6 7 8
Primers
Paired flanking primers Length (17-28bp) GC content 50-60% GC Clamp Tms between 55-80 Avoid simple sequences e.g. strings of Gs Avoid primer self complementary
e.g. hairpins, homodimers, heterodimers
PCR Buffer
Basic Components
20mM Tris-HCL pH 8.4 50mM KCl 1.5 mM MgCl2
Magnesium Since Mg ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many increase the yield of non-specific products and promote misincorporation. Potential Additives
Helix Destabilisers - useful when target DNA is high G/CWith NAs of high (G+C) content.
dimethyl sulphoxide (DMSO), dimethyl formamide (DMF), urea formamide
Long Targets >1kb. Formamide and glycerol Low concentration of template: Polyethylene glycol (PEG)
PCR Polymerases
Taq, Vent, Pfu, others Native or Cloned Half-life
e.g. Taq 40 min half-life, Vent 7 hour half-life
Notes
Typical Reaction
80 l 10 l 2 l 1 l 1 l 5 l 1 l 100 l dH2O 10 X PCR buffer + mg 200 M dNTP 50 M Left Primer 50 M Right Primer Worm Lysate Taq Pol (5 Units/ l) Total Vol
Master Mix
1 Reaction Common Comp
80 l dH2O 10 l 10 X PCR buffer 2 l 200 M dNTP 1 l Taq Pol (5 Units/ l) 93 l Total Volume