User:Karmella Haynes/Notebook/PcTF Genomics/2014/10/08: Difference between revisions
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Samples | |||
# KAH126/MV2, spin | # KAH126/MV2, spin | ||
# mock, spin | # mock, spin | ||
# KAH126/MV2 conventional | # KAH126/MV2 conventional | ||
# mock conventional | # mock conventional | ||
Spinoculation | Spinoculation | ||
* Set up DNA | * Set up Lipo-DNA and mock complexes for 6-well plates (standard Lipo LTX protocol): 2000 μg/ 20 μL DNA, 570 μL Opti-MEM, 2.5 μL PLUS, 7.5 μL Lipofectamine LTX | ||
* Harvest U2OS cells from T-75 flask (~1.0E6 cells/ mL) | * Harvest U2OS cells from T-75 flask (~1.0E6 cells/ mL) | ||
* Transfer ~5.0E5 cells into 15 mL conical | * Transfer ~5.0E5 cells into 15 mL conical. Fill up to final vol. of 3.4 mL with growth medium | ||
* Add 0.6 mL Lipo-DNA or mock complexes. Swirl to mix. | |||
* Spin at 2250 rpm (1000 xg) at room temp for 25 min. | |||
* Transfer most of supernatant into 6-well plate wells | |||
* Flick to disrupt the pelleted cells. Use 1 mL of growth medium to transfer cells to wells. | |||
Conventional | |||
* Remove 0.5 mL growth medium from each well | |||
* Drop-wise, add Lipo-DNA or mock complexes to wells | |||
Grow all cells overnight at 37°C/5% CO2. Check for RFP expression tomorrow | |||
Revision as of 11:50, 8 October 2014
 Pc-TF Genomics
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10/08/14
"Spinoculation" transfection expriment
Conventional
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