User:Karmella Haynes/Notebook/Polycomb project/2010/07/08

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07/08/10
--> Also do RNA prep/ cDNA SYNTH. for 6-well samples (for p16INK RT-PCR)
 * &#x2713; RT-PCR: try p16INK 7B (p16INK f2/r2) on various cDNA samples
 * &#x2713; Western: protein preps from 10 cm and 6-well plates, rotenone -/+ FTRx cells
 * &#x2713; Transfection: puromycin selection plates (25 mL) for KAH130, 131 (dox- samples, 100k and 50k cells), and "no DNA" neg. control (100k cells)
 * &#x2713; Cell culture: split confluent plates 1:10
 * &#x2713; Senesence assay: long term experiment -/+ dox; 6-well plates for KAH126-1, KAH126-3, KAH132-8, KAH154-2, KAH128-8.3, KAH129-4
 * Cytology (prep): 12-well plate for KAH126-1, KAH126-3, KAH132-8, KAH154-2, KAH128-8.3, KAH129-4; DAPI vs. RFP (images for paper) Don't use regular culture plates, use glass-bottom 24-well (#P24G-1.5-13-F) instead
 * &#x2713; Order primers: p16INK 7C (f3/r3); EZH2 (f1/r1); p14ARF 6B (f2/r2); ATF3 (f1/r1); CCND2 (f1/r1)

RT-PCR --> cDNA templates

--> Primer pair: p16INK4a(7B) (make fresh) --> See 4/29/10 for successful p16INK RT-PCR

--> Aliquot 19.5 μL ("1:1" tubes) or 15.0 μL ("1:10" tubes) of each DNA mix to appro. wells --> Add 0.5 (1:1) or 5.0 (1:100) cDNA to each well --> PCR (96-well)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
 * 72°C/ 3 min.
 * 4°C/ ∞

--> Conclusion: Reducing amount of RNA for cDNA synth. appears not to improve specificity of product. DNA contamination? Order new primers based on Agger et al. 2009, where one primer is designed to overlap exon junction (maybe more specific). Also order primers for mRNA's of genes included in ChIP set.

Western: rotenone-treated FTRx

> Protein preps (use RIPA + 1x p.i. cocktail): --> Rotate @ 4°C/ 1 hour --> Spin @ 15,000rpm/ 4°C/ 15 min. Save supernantant @ -20°C until gels arrive. --> Continue with Western tomorrow.
 * 1) 10 cm plate FTRx +DMSO, 500 μL RIPA
 * 2) 10 cm plate FTRx +0.2 μg/mL rotenone, 500 μL RIPA
 * 3) 6-well (1 well) FTRx +DMSO, 200 μL RIPA
 * 4) 6-well (1 well) FTRx +0.2 μg/mL rotenone, 200 μL RIPA

RNA Preps/ cDNA synth.

> RNA Preps --> Use 1 mL TriZol/ well (6-well dishes) --> Resuspend RNA pellet in 10 μL THE RNA Storage Solution

> oligo(dT) Primer annealing

> cDNA synthesis mix --> 10 reactions total

--> Add 10 μL mix to each annealing rxn. --> 50°C/ 50 min., 80°C/ 5 min., ice --> Add 1.0 μL RNase H, 37°C/ 20 min. --> Store at -20°C


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