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

1. prepare the reaction mixture of 50 μL
   1. 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

1. immediately before thermal cycling, aliquot 50 μl of the reaction mixture into a sterile 0.5 mL micro centrifuge tube

1. thermocycle
   1. 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