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

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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05/01/10

 * &#x2713; Cell culture: split KAH126-1, 130-2, 132-8, U2OS plain, Flp-in T-REx 1:10
 * &#x2713; Transfection prep: KAH126-1, 130-2, 132-8 6-well plates for transfections (ab-free)
 * &#x2713; Transfection prep: KAH126-1, 130-2, 132-8 6-well plates for transfections (ab-free)
 * &#x2713; FlpE plasmid transformation

Hygro kill curve > Cells (plate at ~200,000/ well in ~1 mL medium, 24-well plate) > Add hygromycin to the following concentrations...
 * 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)
 * 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 --> 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
 * 1) 1 μL of ~350 ng/μL miniprep (from Bruno) + 10 μL st. dH2O
 * 2) 10 μL st. dH2O

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


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