User:Karmella Haynes/Notebook/Polycomb project/2010/08/26

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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08/26/10

 * &#x2713; Microscopy: immunostaining (overnight)
 * &#x2713; RT-PCR: p16 RT-PCR

Microscopy > Immunostaining: see 8/16/10 for optimization expt. > Cells: KAH130-4, 158-4, 131-4, 159-2 > Antibodies:
 * 1) Histone H3 ab1791 (rabbit)
 * 2) H3K27me3 07-449 (rabbit)
 * 3) H3K9me3 9754 (rabbit)
 * 4) H3K4me3 9751 (rabbit)
 * 5) Bmi1 ab14389 (mouse)
 * 6) PolII 8WG16 (mouse)

RT-PCR > See 8/17/10 for primer optimization expt. > Primer pairs:
 * 1) p16INK 7C (1:1, no dilution)
 * 2) GAPDH 21A (1:1000 dilution)

RT-PCR > cDNA from RNeasy RNA preps 1,2. KAH126-1 -/+ dox 3,4. KAH128-8.3 -/+ dox 5,6. KAH129-4 -/+ dox 7,8. KAH132-8 -/+ dox 9,10. KAH154-2 -/+ dox 11,12. KAH156-5 -/+ dox 13,14. KAH157-1 -/+ dox 15,16. KAH158-4 -/+ dox 17,18. KAH159-1 -/+ dox 19,20. FTRx -/+ dox

--> Aliquot 19.5 μL of each DNA mix to appro. wells --> Add 0.5 (1:1 or 1:1000) cDNA to each well --> PCR (96-well)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32*
 * 72°C/ 3 min.
 * 4°C/ ∞

(* Last time used 35 cycles; this time use 32 to reduce background amplification)


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