AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol

PCR manual

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