User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/23

From OpenWetWare

Jump to: navigation, search
PHIX174 Cell Free Expression Main project page
Previous entry      Next entry

Hypothesis 2: Gene L is necessary for phage propagation.

  • Made new primer 4 and primer 4 T3485A mixes from the source at 100 and 10 μM.
  • 50 μL WP-PCR reaction:
    • 37 μL H2O
    • 5 μL 10X reaction buffer
    • 1.5 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
    • 0.5 μL 25 mM dNTPs mix
    • 1 μL PfuUltra I DNA polymerase
  • Aliquot 2 × 18 μL:
    1. 2 μL 10 μM primer 4 mix
    2. 2 μL 100 μM primer 4 mix
  • Cycling parameters:
    1. 95 °C 2 m
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 72 °C 15 m
    5. Repeat 2-4 an additional 29 times for 30 total cycles
    6. 72 °C 30 m
Personal tools