AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol

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PCR manual

Contents

Materials

  • primers
  • plasmid
  • dexynucleoside triphosphate mixture (dNTPs)

Equipment

PCR tubes temperature cycler

Parameters

  • for DNA vectors (plasmids) of less than 10 kbases)
    • The primers must each be at 100-200 ng/μL
    • 100-250 μM of each dNTPs
    • denaturing temperature is 95 °C
    • extension temperature is 72 °C (extension time 1 min per kb)
    • 25-30 cycles
    • enzyme concentration is 2.5 units of enzyme/ 50 μL of reaction for vector targets up to 19 kb and must be the last component added to PCR mixture (ie after dNTPS) to prevent degradation of the primers.

Protocol

  • prepare the reaction mixture of 50 μL
    • add the components in order

Component Amount per reaction

Component Amount per reaction
Distilled water (dH2O) 40.6 μl
10× cloned Pfu reaction buffer 5.0 μl
dNTPs (25 mM each dNTP) 0.4 μl
DNA template (100 ng/μl) 1.0 μl
Primer #1 (100 ng/μl) 1.0 μl
Primer #2 (100 ng/μl) 1.0 μl
PfuTurbo® DNA polymerase (2.5 U/μl) 1.0 μl (2.5 U)
Total reaction volume 50 μl
  • immediately before thermal cycling, aliquot 50 μl of the reaction mixture into a sterile 0.5 mL micro centrifuge tube
  • thermocycle
    • for targets less than 10 kb vector DNA

1. 2 min at 95 °C
2. repeat this 30 times

   a) 30 s at 95 °C 
b) 30 s at Tm - 5 °C
c) 1 min at 72 oC for each 1 kb

3. 10 min at 72 °C
4. set temperature to 0 °C

Notes

  • Backbone size w/o insert (bp):7328 bp for ADA
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