User:Karmella Haynes/Notebook/Polycomb project/2011/04/20

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04/20/11

  • ✓ qRT-PCR: Pc-TF plates 27, 28
  • ✓ cDNA: HEK-Gal4-luc Pc-TF RNA preps 1-8 (no silencing), 9-15 (2-day dox set, remainder), 17-24 (4-day dox set)
  • ✓ qRT-PCR: HEK-Gal4-luc Pc-TF cDNA plate HEK1



qRT-PCR

> Plate 27
--> 750 nM Primers (& cDNA dilution)

  1. IGFBP2 51A (1:10)

--> Templates, use 2 μL

  1. KAH126-1 (2/21/10)
  2. KAH126-1 +dox "
  3. KAH126-1 (6/15/10)
  4. KAH126-1 +dox "
  5. KAH126-1 (7/26/10)
  6. (no template)
  7. KAH128-8 (2/21/10)
  8. (no template)
  9. KAH128-8.3 (7/26/10)
  10. KAH128-8.3 +dox "
  11. KAH129-4 (2/21/10)
  12. KAH129-4 +dox "
  13. KAH129-4 (7/26/10)
  14. KAH129-4 +dox "
  15. KAH154-2 (6/15/10)
  16. KAH154-2 +dox "
  17. KAH154-2 (7/26/10)
  18. (no template)
  19. KAH154-2 (4/08/11 B)
  20. KAH154-2 +dox "
  21. KAH156-5 (7/26/10)
  22. KAH156-5 +dox "
  23. KAH156-5 (4/08/11 A)
  24. KAH156-5 +dox "
  25. KAH156-5 (4/08/11 B)
  26. KAH156-5 +dox "
  27. KAH157-1 (7/26/10)
  28. KAH157-1 +dox "
  29. KAH157-1 (4/08/11 A)
  30. KAH157-1 +dox "
  31. KAH157-1 (4/08/11 B)
  32. KAH157-1 +dox "

> Plate 25
--> 750 nM Primers (& cDNA dilution)

  1. IGFBP2 51A (1:10)

--> Templates, use 2 μL

  1. KAH132-8 (6/15/10)
  2. KAH132-8 +dox "
  3. KAH132-8 (6/18/10)
  4. KAH132-8 +dox "
  5. KAH132-8 (6/23/10)
  6. KAH132-8 +dox "
  7. FTRx (2/21/10)
  8. FTRx +dox "
  9. FTRx (6/15/10)
  10. FTRx +dox "
  11. FTRx (6/18/10)
  12. FTRx +dox "
  13. FTRx (7/26/10)
  14. FTRx +dox "
Reagent 1 rxn Primer mix (x49), make 2 Primer mix (x43)
diluted cDNA 2.0 --- ---
SYBR Green mix 7.5 367.5 322.5
750 nM primers 3.0 147.0 129.0
dH2O 2.5 122.5 107.5
  15.0

--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) DNA to 39.0 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> 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



cDNA synthesis
--> Samples: Cells treated with dox for 0, 2, 4 days; transfected with Pc-TF/MV1 constructs 5 days after dox wash-out

Sample A260 260/280 ng/ul
1. 0 dox/ 160 1.461 1.74 58.4
2. 0 dox/ 165 1.215 1.56 48.6
3. 0 dox/ 161 0.964 1.64 38.6
4. 0 dox/ 167 0.995 1.61 39.8
5. 0 dox/ 162 1.677 1.62 67.1
6. 0 dox/ 166 1.159 1.67 46.3
7. 0 dox/ 170 1.283 1.76 51.3
8. 0 dox/ mock 1.359 1.77 54.4
9. 2dy dox/ 160 1.662 1.62 66.5
10. 2dy dox/ 165 1.282 1.54 51.3
11. 2dy dox/ 161 1.289 1.56 51.6
12. 2dy dox/ 167 1.387 1.67 55.5
13. 2dy dox/ 162 1.157 1.64 46.3
14. 2dy dox/ 166 1.156 1.70 46.2
15. 2dy dox/ 170 3.624 1.89 145.0
16. done
17. 4dy dox/ 160 4.388 1.90 175.5
18. 4dy dox/ 165 4.293 1.89 171.7
19. 4dy dox/ 161 4.907 1.87 196.3
20. 4dy dox/ 167 4.156 1.88 166.3
21. 4dy dox/ 162 3.799 1.90 152.0
22. 4dy dox/ 166 4.175 1.90 167.0
23. 4dy dox/ 170 4.421 1.90 176.8
24. 4dy dox/ mock 4.987 1.92 199.5


> oligo(dT) Primer annealing
--> Do 4 rxn's for 129-4 #1
--> 2 rxn's ea. for 129-4 +dox #4 & #5

Reagent Vol
total RNA (up to 2μg) 8.0
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 (x23)
10x RT buffer 2.0 46.0
25 mM MgCl2 4.0 92.0
0.1 M DDT 2.0 46.0
RNaseOUT 1.0 23.0
SuperScript III RT 1.0 23.0
  10.0 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



qRT-PCR

> Plate HEK1
--> 750 nM Primers (& cDNA dilution)

  1. GAPDH 21A (1:100)
  2. mCh1 "
  3. luc1 "
  4. luc4 "

--> Templates, use 2 μL

  1. (17) 4dy dox/ 160
  2. (18) 4dy dox/ 165
  3. (19) 4dy dox/ 161
  4. (20) 4dy dox/ 167
  5. (21) 4dy dox/ 162
  6. (22) 4dy dox/ 166
  7. (23) 4dy dox/ 170
  8. (24) 4dy dox/ mock


Reagent 1 rxn Primer mix (x25)
diluted cDNA 2.0 ---
SYBR Green mix 7.5 187.5
750 nM primers 3.0 75.0
dH2O 2.5 62.5
  15.0

--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) DNA to 39.0 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> 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