User:Karmella Haynes/Notebook/miR Trigger/2009/12/22

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12/22/09

 * &#x2713; Minipreps: KAH93-95/MV2 (2 each)
 * &#x2713; Transfection 1: KAH93, 94, 95/MV2 for microscopy and RT-PCR (6-well, Lipo)
 * &#x2713; Transfection 2: KAH94, 95/MV2 for miRNA dose response curve (24-well, Fugene)
 * &#x2713; Re-transform plasmids: KAH93, 94, 95/MV2 ( Did not work well, re-try with Z-DH5α, 12/23/09 &#x2713; )

Minipreps > Check with E/P digests

> No plasmid DNA. Re-transform from minipreps (quick & dirty transformation, DH5α T1R lab stock)

Transfection 1 > Details

> Add 2x Lipo to 500 μL Opti-MEM --> R.T/ 5 min. > Add 2x DNA to 500 μL Opti-MEM > Add DNA mix to Lipo mix --> R.T./ 20 min. > Remove 500 μL medium from each well; Add 500 μL complexes to each well (4 ml total vol.); Grow cells at 37&deg;C > Refresh medium after 5 hours (ab-free)

Day 2 > Add 1μg/mL Dox to Dox+ samples. Incubate overnight (~20 hours).

Day 3 > RNA preps/ cDNA synthesis

Transfection 2 > Details

> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min. > Add 6x vol DNA --> R.T./ 20 min. > Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37&deg;C overnight

Day 2 > Refresh w/ ab-free, no phenol red medium > Add varying doses of doxycycline to the wells in each row: 0, 0.1, 1.0, 10.0, 100.0, 1000.0 ng/mL ---> 1:10 serial dilutions; dilute dox in 50 μL medium > Assay RFP/YFP after 5 or 6 hours (plate reader) --> Accidentally did 1:100 serial dilution (0, 0.00001, 0.001, 0.1, 10.0, 1000.0) for Read #1 --> Refreshed medium and used correct 1:10 dilutions of Dox for overnight induction > Grow overnight

Day 3 > Read #2: Assay RFP/YFP (morning)


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