User:Karmella Haynes/Notebook/Polycomb project/2011/03/17

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03/17/11

  • ✓ qRT-PCR: Pc-TF -/+ dox, plate 5, 6
  • ✓ RT-PCR: optimize more primers with KAH126-1 +dox cDNA


qRT-PCR

> Plate 5
--> 750 nM Primers (& cDNA dilution)

  1. mCh1 (1:1000)
  2. GAPDH 21A (1:1000)
  3. ACTC1 35A (1:10)
  4. GRLH2 37C (1:10)

--> Templates, use 2 μL

  1. KAH126-1, 2/21/10
  2. KAH126-1 +dox, 2/21/10
  3. KAH128-8, 2/21/10
  4. KAH128-8 +dox, 2/21/10
  5. KAH129-4, 2/21/10
  6. KAH129-4 +dox, 2/21/10
  7. FTRx, 7/26/10
  8. FTRx +dox, 7/26/10


> Plate 6
--> 750 nM Primers (& cDNA dilution)

  1. NPPA 40A (1:10)
  2. CDKN2A 7C (1:10)
  3. MMP12 31B (1:10)
  4. THPO 33A (1:10)

--> Templates (same), use 2 μL

  1. KAH126-1, 2/21/10
  2. KAH126-1 +dox, 2/21/10
  3. KAH128-8, 2/21/10
  4. KAH128-8 +dox, 2/21/10
  5. KAH129-4, 2/21/10
  6. KAH129-4 +dox, 2/21/10
  7. FTRx, 7/26/10
  8. FTRx +dox, 7/26/10


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


RT-PCR
> Test new RT-PCR primers on KAH126-1 (36 rxns total)
> Primers:
1-4. CASP10 41A-D (80, 84, 80, 93 bp)
5-8. EOMES 42A-D (101, 106, 102, 120 bp)
9-12. OTX2 43A-D (102, 108, 105, 88 bp)
13-16. PRKACG 44A-D (88, 99, 90, 86 bp)
17-20. SST 45A-D (106, 82, 102, 81 bp)
21-24. ATOH1 46A-D (119, 116, 93, 100 bp)
25-28. HHEX 47A-D (86, 80, 87, 113 bp)
29-32. IL4 48A-D (97, 102, 106, 94 bp)
33-36. SLC6A4 49A-D (84, 86, 80, 83 bp)

> Template:

  1. KAH126-1 -dox 1:1


Reagent 1 rxn Template mix (37x)
cDNA 0.5 18.5
10 μM primers 1.0 ---
2x GoTaq Green 10.0 370.0
dH2O 8.5 314.5
  20.0 μL

--> 1 template mix: aliquot 19.0 μL
--> Add 1.0 μL primers

--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 15 sec., 58°C/ 15 sec., 72°C/ 15 sec.] x32
  • 72°C/ 3 min.
  • 4°C/ ∞


RT-PCR 3/17/11

--> Conclusions:

  1. CASP10: qPCR test 41A, D
  2. EOMES: qPCR test 42A, D
  3. OTX2: qPCR test 43D
  4. PRKACG: too non-specific
  5. SST: qPCR test 45C, D
  6. ATOH1: qPCR test 46A, D
  7. HHEX: failed
  8. IL4: failed
  9. SLC6A4: non-specific (49C)

Next: Do qRT-PCR test with 1:10, 1:100 dilutions of 126-1 (+dox bulk cDNA)



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