User:Karmella Haynes/Notebook/Polycomb project/2010/05/23

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05/23/10

 * &#x2713; Growth assay: cell count (day 6); split stock cultures (1x 10 cm plain medium, 1x 10 cm +antibiotics per line; 1:10)
 * &#x2713; Western blot: Pc-ATF negative ctrl lines

Western blot

> SDS-PAGE --> Pc-ATF negative ctrl lines --> 4x 10-well tris-glycine (4-20%) pre-cast gels --> Samples (25 μL total vol./ well); use 15 μL PageRuler Plus pre-stained ladder --> Make 100 mM DTT in 2x loading buffer (> enough for 12.5 μL/ gel lane) --> Add 12.5 μL DTT + buffer to each protein sample --> Incubate at 100°C/ 5 min. --> Run gel @ 120 V until dye front reaches the bottom

> Electroblot --> Run @ 100 V for 1 hr.

--> Blot #4 messed up, forgot to add top sponge to blot; Do over

> Immunostaining --> Block 4°C/ overnight; continue Mon.

5/24/10 --> Primary (4°C/ overnight):
 * 1) rabbit anti-myc (ab1791), 1:4000, 10 mL

5/25/10 --> Secondary: donkey anti-rabbit IgG-HRP, 1:5000 (R.T./ 1 hr.) (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)

--> Oops! Mixed up a couple of samples: 33. 159-2 34. 159-3 35. 159-3 +dox 36. 159-2 +dox

> Conclusions: Use the following lines in further experiments
 * 1) KAH154-2 (hPcCD/-VP64)
 * 2) KAH155-2 (mPcCD/-VP64)
 * 3) KAH156-5 (fshPcCD/-VP64)
 * 4) KAH157-1 (flyPcCD/-VP64)
 * 5) KAH158-4 (hCBX8/-VP64)
 * 6) KAH159-2 (flyPc/-VP64)

Growth assay (day 6) > Do cell counts for the following: --> Omitted KAH126-4; no RFP detected on day 4; lost RFP expression? > Trypsinize cells with 200 μL Trypsin buffer > Resuspend cells with 800 μL medium (1 mL total volume) > Dilute samples 1/4 for Scepter counting; add 25 μL cells to 75 μL PBS
 * 1) KAH126-1
 * 2) KAH126-3
 * 3) KAH132-8
 * 4) FTRx
 * 5) KAH126-1 +dox
 * 6) KAH126-3 +dox
 * 7) KAH132-8 +dox
 * 8) FTRx +dox


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