User:Karmella Haynes/Notebook/Polycomb project/2010/12/16

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12/16/10

  • Postpone Pc-TF histone peptide pull-down; switch priority to follow-up on senescence
  • ✓ Culture: Thaw 126-3, 154-2, 132-8, FTRx for growth assays
  • ✓ Growth assay optimization: plate 126-1 (PS1) in 6-well plates; make 1:10 back-up

--> Plan: follow single colonies over time, count cells over time; include in B-gal data figure

  • ✓ ChIP: chromatin IP overnight

--> Note: tomorrow, chromatin preps for 128-8.3 and 129-4 fx cells



Growth assay
> Do growth assay by tracking single colonies over time
> Optimize assay using KAH126-1 > Plates

  1. - dox
  2. - dox
  3. + 1.0 ug/mL dox
  4. + 1.0 ug/mL dox

> Wells:
1,2. ~2x10^5 (5x dilution of 1x10^6 suspension from confluent flask)
3,4. ~1.0x10^5
5,6. ~5.0x10^4
7,8. ~2.5x10^4
9,10. ~1.26x10^4
11,12. ~6.25x10^3

> Will pick best dilution range for future assays



ChIP
> Lack of DNA IP might be problem due to non-saturating amounts of beads; lots of the over-expressed Pc-TF protein may not be bound.
> Myc/IgG-bead IP: Use 30 uL beads/ 500 mL chromatin (used only 10 uL before)
> H3K27me3/IgG IP: Use 5 uL ab/ 500 mL chromatin (used only 2 uL on 11/30/10) > Samples (all form. x-linked chromatin)
1/2. 126-1: + 30 μL αmyc-beads (3400); + 30 μL mouse IgG-beads (3420)
3/4. 130-4: "
5/6. 132-8: "
7/8. FTRx: + 5 μL αH3K27me3 (07-449); + 5 μL rabbit IgG (sc-2027)
> Rotate at 4°C/ overnight (will add beads to 7/8 tomorrow morning)