User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/20

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05/20/15

  • Cas-tone - Phusion PCR for H3


Cas-tone - Phusion PCR for H3

  • Phusion PCR-amplify promoters
  1. pAubR, EcoRI fwd, SpeI rev
  2. pBjaR, EcoRI fwd, SpeI rev
  3. pBraR, EcoRI fwd, SpeI rev
  4. pRpaR, EcoRI fwd, SpeI rev
Reagent Vol Mix (x6) Expected:
1. pAubR = 153
2. pBjaR = 122
3. pBraR = 214
4. pRpaR = 136 		Hover name
5 μL/lane; 1% agarose; Ladder
gBlock DNA (2 ng/μL) 0.5 3.0
10 μM H3_1 f1 1.0 6.0
10 μM r primer 1.0 ---
10 mM dNTPs 1.0 6.0
5x HF buffer 10.0 60.0
Phusion Pol. 0.5 3.0
dH2O 36.0 216.0
  50.0 μL

Thermal cycler: Bio-Rad - Phusion

  • 98°C 3 min.
  • 30x[98°C, 10 sec; 70°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞





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