User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/29

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01/29/10

  • ✓ SOEing PCR: repeat megaprimer PCR from 1/28
  • ✓ Cultures: KAH139/V0201 (4)

> Alternative Step 2
--> Repeat "megaprimer" PCR from 1/28; 4 reactions

Reagent Volume Expected:
1184 bp
PCR 1/28/10
10 μL/lane; 1% agarose
DNA(plasmid) 0.5 (from Bruno)
amplicon 1 (clean) 2.0
amplicon 2b (clean) 2.0
2x GoTaq green 25.0
dH2O 20.5
  50 μL

--> BioRad Block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞

--> PCR didn't work. Try site-directed mutagenesis tomorrow.



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