User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/29: Difference between revisions

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* Trasformation
* Trasformation
** KAH87 is running low.
** KAH87 is running low.
** Retransform into Dh5α-turbo cells: 0.5 μL plasmid, 9.5 μL dH<sub>2</sub>O (quick protocol)
** Retransform KAH87 and MV10 into Dh5α-turbo cells: 0.5 μL plasmid, 9.5 μL dH<sub>2</sub>O (quick protocol)
** Plate on 100 μg/mL amp
** Plate on 100 μg/mL amp


Revision as of 10:37, 1 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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




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