User:Karmella Haynes/Notebook/Polycomb project/2010/09/13

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09/13/10

  • ✓ RNA prep/ cDNA: p16 induction time course
  • ✓ ChIP/ Co-IP: KAH130-4 sample
  • ✓ Midipreps: Use QIAfilter Plasmid Midi kit (100 mL culture, used Maxi volumes to prep and ppt lysate, midi volumes to wash/ elute/ ppt DNA)
  • ✓ Culture: HEK cells failed to grow; get back-up vial from Mirra



RNA prep
> Use Qiagen QIAshredder and RNeasy kit (no BME added to lysis buffer)
> Elute with 50 μL RNase-free H2O

Sample A260 260/280 ng/ul uL = 2ug RNA dH2O
1. KAH126-1, 1-day 17.289 2.08 691.58 2.9 5.1
2. KAH126-1, 2-day 16.842 2.12 673.67 3.0 5.0
3. KAH126-1, 3-day 17.923 2.06 716.92 2.8 5.2
4. KAH126-1, 4-day 15.9 2.05 635.99 3.1 4.9
5. KAH154-2, 1-day 23.301 2.07 932.03 2.1 5.9
6. KAH154-2, 2-day 16.135 2.09 645.41 3.1 4.9
7. KAH154-2, 3-day 18.431 2.08 737.24 2.7 5.3
8. KAH154-2, 4-day 13.603 2.08 544.11 3.7 4.3
9. KAH132-8, 1-day 18.812 2.07 752.49 2.7 5.3
10. KAH132-8, 2-day 15.47 2.06 618.8 3.2 4.8
11. KAH132-8, 3-day 13.727 2.08 549.07 3.6 4.4
12. KAH132-8, 4-day 10.627 2.01 425.07 4.7 3.3
13. FTRx, 1-day 20.22 2.07 808.8 2.5 5.5
14. FTRx, 2-day 22.792 2.08 911.69 2.2 5.8
15. FTRx, 3-day 22.614 2.09 904.55 2.2 5.8
16. FTRx, 4-day 14.333 2.07 573.32 3.5 4.5

--> Store remainder of RNA at -80°C


7/26/10
> oligo(dT) Primer annealing

Reagent Vol
total RNA (up to 2μg) up to 8 μL
50μM oligo(dT) primer 1.0
10 mM dNTP mix 1.0
DEPC-treated water ---
  10.0 μL --> 65°C/ 5 min.; ice/ 1 min.


> cDNA synthesis mix
--> 16 reactions total

Reagent Vol Mix (x16)
10x RT buffer 2.0 32.0
25 mM MgCl2 4.0 64.0
0.1 M DDT 2.0 32.0
RNaseOUT 1.0 16.0
SuperScript III RT 1.0 16.0
  10.0 μL 160.0 μL

--> 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



ChIP: myc-bead pull-down
> See 6/29/10
> Prep sonicated ~6 mL samples according to Qingqing's protocol
--> Add:

  • 720 μL 10% Triton X-100
  • 72 μL 10% Na-deoxycholate (DOC)
  • 500 μL TE (pH 8.0)
  • 6 μL 1000x PLAAC
  • 60 μL 100x PMSF

--> Save 4x 100 μL samples as "Input"

> Binding:

  1. KAH130-4: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  2. KAH130-4: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

--> Rotate at 4°C overnight
--> Wash and elute tomorrow


9/14/10
> Wash & elute IP's (Keep everything on ice)
--> Prepare washing buffer (complete sonication buffer):

  • 6 mL Buffer III
  • 60 μL 100x PMSF
  • 6 μL 1000x PLAAC
  • 720 μL 10% Triton X-100
  • 72 μL 10% Na-deoxycholate (DOC)
  • 500 μL TE (pH 8.0)

--> Spin down beads at 3000 rpm/ 3 min./ 4°C
--> Save 50 μL supernatant. Discard remaining sup.
--> Wash beads in 500 μL wash buffer (1 min.). Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup. Repeat three more times (4 washes total)
--> Add 60 μL 1x loading dye (50 uL 4x l.d. + 150 RIPA, ~50 mM DTT) to each pellet. Heat at 100°C/ 5 min., vortex.
--> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube (save at -20°C for Western later).



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