User:Karmella Haynes/Notebook/Polycomb project/2009/12/01

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12/01/09

  • Transfections: mCh-VP64 (trial 3)

Transfections
> HEK293 cells + Gal4/mCh/VP64 constructs
> Use Fugene, 24-well format
> Serial dilutions of plasmids
--> Start with 1600 ng in 10 μL OptiMEM

Plasmid ng/μL Add (μL)... to OptiMEM (μL)
1. KAH59/VNpc 356.0 4.5 5.5
2. KAH60/VNpc 405.2 3.9 6.1
3. KAH54/VNpc 464.4 3.4 6.6
4. KAH66/MV5 429.4 3.7 6.3
5. KAH65/MV5 413.7 3.9 6.1
6. KAH59/MV5 427.6 3.7 6.3
7. KAH36 778.5 2.1 7.9

--> Add 5 μL plasmid to 5 μL OptiMEM for each serial dilution

Wells Plasmid DNA Volume Fugene Opti-MEM
1-6 KAH59/VNpc 25, 50, 100, 200, 400, 800 ng 5.0 μL 1.8 μL 18.2 μL
7-12 KAH60/VNpc " " " "
13-18 KAH54/VNpc " " " "
19-24 KAH66/MV5 " " " "
25-30 KAH65/MV5 " " " "
31-36 KAH59/MV5 " " " "
37-42 KAH36 " " " "
43-48 none " " " "

> Master mix: 90 μL Fugene + 910 μL Opti-MEM --> R.T/ 5 min.
> Add 20 μL Fugene mix to DNA --> R.T./ 20 min.
> Add 25 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay RFP/ Luc after 18-24 hours



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