User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/29

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04/29/15

  • LCR - Phusion PCR for MV10
  • KAH126 expression - rebuild PcTF expression plasmid for K562



MV10 Phusion PCR

  • Use linearized MV10 (XbaI-cut)
Reagent Rxn1 Rxn2 Expected:
1,2. MV10 = 5191
Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.5 0.5
10 uM fwd primer 1.0 1.0
10 uM rev primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion pol. 0.5 0.5
5x buffer 10.0 HF 10.0 GC
DMSO --- 1.5
dH2O 36.0 34.5
  50.0 50.0

Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 3.0 min]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • No bands. Try again later with fresh template



KAH126 expression - rebuild PcTF expression plasmid for K562

  • Insert KAH87 (X/S) into MV10 (X)
  • KAH87 = hPCD : [mCh : VP64]


  • Trasformation
    • KAH87 is running low.
    • Retransform KAH87 and MV10 into Dh5α-turbo cells: 0.5 μL plasmid, 9.5 μL dH2O (quick protocol)
    • Plate on 100 μg/mL amp