User:Karmella Haynes/Notebook/Polycomb project/2010/09/15

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09/15/10

 * &#x2713; Western: KAH130-4 IP samples
 * &#x2713; RT-PCR: test new primers for Nanostring gene candidates
 * &#x2713; Cell culture: thaw HEK Gal4-EED line (from Mirra)
 * &#x2713; ChIP: IP for KAH126-1, 130-2 (double x-linked), and 130-4 (single x-linked) for DNA isolation
 * &#x2713; RT-PCR: Nanostring gene candidates IL4, MMP12, THPO, TNF on full cDNA set

Western > IP samples from 9/13/10; already prepped for loading (2 wells worth) > Prep input and sup samples: 22.5 protein + 7.5 4x loading dye --> 4x loading dye (Invitrogen) w/ freshly added DTT (200 mM final) > Use 10-well gel (loading volume = 25 μL) > Electroblot: 1 hr. 15 min.

> Block: 5% milk/PBST, R.T./1 hr.

> Primary staining: 5% BSA/PBST, 4°C/o.n.
 * 1) rabbit α-DsRed 632496, 1:1000, 5 mL
 * 2) rabbit α-H3K27me3 07-449, 1:500, 5 mL

8/28/10 > Secondary staining: 5% milk/PBST, R.T./ 1 hr. > Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)
 * 1) donkey α-rabbit-HRP, 1:5000; 5 mL
 * 2) donkey α-rabbit-HRP, 1:5000; 5 mL
 * KAH130-4: 79 kD
 * H3K27me3: 15 - 17 kD

Conclusions:
 * Blot 1: 130-4 pull-down looks perfect. Very clean and specific.
 * Blot 2: 17 kD H3K27me3 shows up at right size in input and sup lanes, but what is the mysterious 28 kD band? Same as observed before with DMA/formaldehyde cross-linked KAH126-1 & 132-8 (8/28/10). Could be something coming of of the α-myc beads during elution that is recognized by α-H3K27me3 primary or α-rabbit secondary. No 17 kD band. Try total IP next time (and myc-bead/ no chrom control?).

RT-PCR > Use cDNA from Nanostring set > Primers + NS Templates:
 * 1) HHEX 28C (126 bp) + KAH129-4 -/+ dox (2 rxns)
 * 2) IL4 30A (107 bp) + KAH129-4 -/+ dox (2 rxns)
 * 3) IL4 30B (89 bp) + KAH129-4 -/+ dox (2 rxns)
 * 4) MMP12 31A (127 bp) + KAH126-1 -/+ dox, KAH128-8 -/+ dox, KAH129-4 -/+ dox (6 rxns)
 * 5) MMP12 31b (99 bp) + KAH126-1 -/+ dox, KAH128-8 -/+ dox, KAH129-4 -/+ dox (6 rxns)
 * 6) NPPA 32A (99 bp) + KAH126-1 -/+ dox (2 rxns)
 * 7) NPPA 32B (99 bp) + KAH126-1 -/+ dox (2 rxns)
 * 8) THPO 33A (103 bp) + KAH126-1 -/+ dox, KAH128-8 -/+ dox (4 rxns)
 * 9) THPO 33B (105 bp) + KAH126-1 -/+ dox, KAH128-8 -/+ dox (4 rxns)
 * 10) TNF 34A (119 bp) + KAH128-8 -/+ dox (2 rxns)
 * 11) TNF 34B (107 bp) + KAH128-8 -/+ dox (2 rxns)

--> 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/ ∞



> Conclusions:
 * MMP12, THPO, and TNF successfully detected. Repeat PCR for whole cDNA set, use fewer cycles (32, same as p16).
 * IL4 worked somewhat; a little non-specific. Try fewer cycles.
 * HHEX shows very faint bands, but product appears to be the right size.
 * NPPA failed. Order new primers.

RT-PCR > Use cDNA from Nanostring set (omit KAH158, 159) > Primers + NS Templates:
 * 1) IL4 30B (89 bp) + NS cDNA (16 rxns)
 * 2) MMP12 31B (99 bp) + NS cDNA (16 rxns)
 * 3) THPO 33A (103 bp) + NS cDNA (16 rxns)
 * 4) TNF 34A (119 bp) + NS cDNA (16 rxns)

--> 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/ ∞



> Conclusions:
 * IL4 too non-specific. Do not see induction in specifically in 129-4
 * MMP12 looks good, but has ns lower band. Try again with 1:10 dilution of cDNA and primer pair 31A
 * THPO, hard to see differences. Try again with 1:10 dilution of cDNA
 * TNF looks great. Use data for figure.
 * Try the successful primers on previous biological replicates.

ChIP: myc-bead pull-down > See 6/29/10 > Sonicated ~6 mL samples previously prepped 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

> Binding: --> Rotate at 4°C overnight --> Wash and elute tomorrow
 * 1) KAH126-1: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 2) KAH126-1: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
 * 3) KAH132-8: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 4) KAH132-8: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
 * 5) KAH130-4: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 6) KAH130-4: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

9/14/10 > Wash & elute IP's (Keep everything on ice ) --> Prepare washing buffer (complete sonication buffer): --> Spin down beads at 3000 rpm/ 3 min./ 4°C --> Save 100 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 500 μL 1% SDS/TE buffer. Incubate at 65°C/ 15 min. Vortex every 2 min. --> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube.
 * 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)

> Reverse crosslinks and digest protein --> Add 400 uL 1% SDS/TE buffer to 100 uL saved Supernatant. --> Thaw Input aliquot for each cell line. Add 400 uL 1% SDS/TE buffer to 100 uL Input. --> Samples:
 * 1) KAH126-1 input
 * 2) KAH126-1 α-myc Sup
 * 3) KAH126-1 α-myc IP
 * 4) KAH126-1 α-IgG Sup
 * 5) KAH126-1 α-IgG IP
 * 6) KAH130-4 input
 * 7) KAH130-4 α-myc Sup
 * 8) KAH130-4 α-myc IP
 * 9) KAH130-4 α-IgG Sup
 * 10) KAH130-4 α-IgG IP
 * 11) KAH132-8 input
 * 12) KAH132-8 α-myc Sup
 * 13) KAH132-8 α-myc IP
 * 14) KAH132-8 α-IgG Sup
 * 15) KAH132-8 α-IgG IP

--> Add 20 μL (20 μg) Pronase to each sample --> Incubate at 42°C/ 1 hr. --> Incubate at 65 °C overnight


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