User:Karmella Haynes/Notebook/Polycomb project/2011/04/18

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04/18/11

  • ✓ ChIP qPCR: Pc-TF plates ch2, ch3
  • ✓ Transfection: repeat 4/12/11 expt. (11 days after dox w/o)
  • ✓ PCR: test luc primers



ChIP qPCR
> See 12/20/11
> Set up each reaction 4x

>Plate ch2
--> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):

  • 128-8.3 (40) input, pos
  • 128-8.3 (41) myc IP, uk
  • 128-8.3 (42) IgG IP, neg
  • 129-4 (43) input, pos
  • 129-4 (44) myc IP, uk
  • 129-4 (45) IgG IP, neg

--> 750 nM primers (24 rxns per primer pair):

  1. ATOH1 A1
  2. ATOH1 B3
  3. ATOH1 C2
  4. ATOH1 D1


>Plate ch3
--> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):

  • FTRx (29) input, pos
  • FTRx (38) myc IP, uk
  • FTRx (39) IgG IP, neg

--> 750 nM primers (12 rxns per primer pair):

  1. ATOH1 A1
  2. ATOH1 B3
  3. ATOH1 C2
  4. ATOH1 D1


Reagent 1 rxn Primer mix (x25) Primer mix (x13)
ChIP DNA 2.0 --- ---
SYBR Green mix 7.5 187.5 97.5
750 nM primers 3.0 75.0 39.0
dH2O 2.5 62.5 32.5
  15.0

--> Aliquot 52.0 primer mix into 1st well of each 4x set
--> Add 8.0 (2.0 x4) DNA to 52.0 primer mix
--> Aliquot 15.0 rxn mix to other 3 wells in each 4x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> Note: Use increased annealing temp compared to previous ChIP PCR's (new primers optimized for 58°C) --> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film

  • 95°C/ 5 min.
  • [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 58°C -> 95°C/ 0.5°C per step



Fugene transfection

  1. KAH160: human Pc-TF
  2. KAH165: human PCD
  3. KAH161: fly Pc-TF
  4. KAH167: fly PCD
  5. KAH162: fish Pc-TF
  6. KAH166: fish PCD
  7. KAH170: deleted PCD
  8. mock (Fugene)


--> Plate 1a/b: no dox
--> Plate 2a/b: Dox-treated cells (4 days, wash-out day 11)
--> Note: setting up double plates to have enough cells for RNA preps

> Plate cells: Cells are very confluent; Collect cells from 1 10cm plate, resuspend 1/2 of cells in 25 mL medium, aliquot 1 mL per well in 24-well plate
--> Also split back-up plates 1:4 to make 4 per treatment; discard extras


Wells Plasmid DNA Volume Fugene Opti-MEM
1-3 KAH160/MV1 100, 200, 400 ng 1.2 μL = 400 ng 1.8 μL 18.2 μL
4-6 KAH165/MV1 " 0.8 μL = 400 ng " "
7-9 KAH161/MV1 " 1.0 μL = 400 ng " "
10-12 KAH167/MV1 " 1.0 μL = 400 ng " "
13-15 KAH162/MV1 " 1.1 μL = 400 ng " "
16-18 KAH166/MV1 " 0.9 μL = 400 ng " "
19-21 KAH170/MV1 " 1.0 μL = 400 ng " "
22-24 mock " 0 " "

> Add DNA to stdH2O for a total volume of 5 μL (4x master mix = 20 μL)
--> Serial dilutions: start w/ 8x DNA + dH2O = 40 μL --> Use 20 μL for serial dilutions
> Fugene master mixes (x4, 24 total): 7.2 μL Fugene + 72.8 μL Opti-MEM --> R.T/ 5 min.
> Add 20 μL DNA mix to each Fugene mix --> R.T./ 20 min.
> Add 25 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay luc activity after 2 days



PCR: test luc primers
> Test new luc primers on HEK-luc cDNA
> Templates (5 rxns ea.)

  1. 2/mock (#16) 1:10
  2. 2/mock (#16) 1:100
  3. 2/mock (#16) 1:1000

> Primers (10 μM)

  1. luc 1 (110 bp)
  2. luc 2 (98 bp)
  3. luc 3 (117 bp)
  4. luc 4 (105 bp)
  5. GAPDH 21A (100 bp)


Reagent 1 rxn cDNA mix (x6)
cDNA 1.0 6.0
2x GoTaq Green 10.0 60.0
10 μM primers 1.0 ---
dH2O 8.0 48.0
  20.0


Bio-RAD 96-well

  • 95°C/ 3 min.
  • [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x30
  • 72°C/ 3 min.

File:KAH 041811 gel1.tif

> Conclusions: Primer pairs luc1, luc2, and luc4 work well. Use 1:100 dilution for subsequent qRT-PCR.