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

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:

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