User:Madeline Hartman/Notebook/Dr. Hartings Lab/2012/11/06

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Entry title

I ran two PCR reactions with the following content:

Tube 1-

  1. 40.6 μL distilled H2O
  2. 5.0 μL 10x cloned Pfu buffer
  3. 0.4 μL dNTPs (25 mM each)
  4. 1.0 μL DNA template (100 ng/μL)
  5. 1.0 μL Primer 1 (ng/μL)
  6. 1.0 μL Primer 2 (ng/μL)
  7. 1.0 μL Pfu Turbo DNA Polymerase (2.5 U/μL)

and

Tube 2-

  1. 20.3 μL distilled H2O
  2. 5.0 μL 10x cloned Pfu buffer
  3. 0.4 μL dNTPs (25 mM each)
  4. 1.0 μL DNA template (100 ng/μL)
  5. 1.0 μL Primer 1 (ng/μL)
  6. 1.0 μL Pfu Turbo DNA Polymerase (2.5 U/μL)

Tube 3-

  1. 20.3 μL distilled H2O
  2. 5.0 μL 10x cloned Pfu buffer
  3. 0.4 μL dNTPs (25 mM each)
  4. 1.0 μL DNA template (100 ng/μL)
  5. 1.0 μL Primer 2 (ng/μL)
  6. 1.0 μL Pfu Turbo DNA Polymerase (2.5 U/μL)

Tubes 2 and 3 were later combined.

Primer 1- t109c forward

Primer 2- t109c reverse

The three tubes were ran for the following cycles:

MH 20121106Table of cycles.png


After 5 cycles, tubes 2 and 3 were combined, and run the rest of the cycles together.

Tubes were left in thermocycler overnight at 4°C