User:Karmella Haynes/Notebook/Polycomb project/2011/03/10

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

03/10/11

 * &#x2713; RNA prep/ cDNA: KAH126 -/+ dox
 * &#x2713; Luc activity assay: luc repressed 6-day, Pc-TF 4-day

RNA prep > Use TriZOL > Dissolve each pellet in 15 μL THE RNA Storage Sln.

Note: *Accidentally added phenol:chloroform:isoamyl alcohol to cells (sample #1)

> oligo(dT) Primer annealing --> Set up four reactions for each condition:
 * 1) Dox-: use sample #3
 * 2) Dox+: use sample #6

> cDNA synthesis mix --> 16 reactions total

--> Add 10 μL mix to each annealing rxn. --> 50°C/ 50 min., 80°C/ 5 min., ice --> Add 0.8 μL RNase H, 37°C/ 20 min. --> Store at -20°C

Luc Activity Assay

Luc activity assay > Cells: dox-induced Gal4-EED expression/ 2 days, followed by transfection for 4 days 1-6. KAH160/MV1 (human Pc-TF), 0, 50, 100, 200, 400, 800 ng plasmid 7-12. KAH170/MV1 (human Pc-TF), 0, 50, 100, 200, 400, 800 ng plasmid > Sample processing:
 * Add 500 μL medium to cells in rep 1 set (measured before); Resuspend cells in 1 mL medium total
 * Aliquot 3x 100 μL samples into 96-well plate
 * Save the rest for cell count (Millipore Scepter)
 * Normalize each value to cell count


 * }