User:Karmella Haynes/Notebook/Polycomb project/2010/05/01

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05/01/10

  • ✓ Cell culture: split KAH126-1, 130-2, 132-8, U2OS plain, Flp-in T-REx 1:10
  • ✓ Transfection prep: KAH126-1, 130-2, 132-8 6-well plates for transfections (ab-free)
  • ✓ Transfection prep: KAH126-1, 130-2, 132-8 6-well plates for transfections (ab-free)
  • ✓ FlpE plasmid transformation



Hygro kill curve
> Cells (plate at ~200,000/ well in ~1 mL medium, 24-well plate)

  1. Flp-in T-REx (blast, zeo)
  2. KAH126-1 (puro, blast, zeo)
  3. KAH130-2 (puro, blast, zeo)
  4. KAH132-8 (puro, blast, zeo)

> Add hygromycin to the following concentrations...

  1. 0 μg/mL
  2. 50 μg/mL
  3. 100 μg/mL
  4. 200 μg/mL
  5. 300 μg/mL
  6. 400 μg/mL



FlpE plasmid transformation

  1. 1 μL of ~350 ng/μL miniprep (from Bruno) + 10 μL st. dH2O
  2. 10 μL st. dH2O

--> Add to 50 μL T1R DH5α
--> Heat shock @ 42°C/ 90 sec
--> Recover @ 37°C/ 30 min.
--> Plate on 20 μg/mL amp (spread amp onto plain LB agar plates = ~25 mL agar)
--> 37°C/ overnight


--> 5/03/10: 3 colonies on transformation plate, 0 on neg. ctrl. Picj 3 colonies