04/15/11
- ✓ qRT-PCR: Pc-TF -/+ dox plates 23, 24
- ✓ RNA prep/ cDNA: Try TRIzol/ RNeasy combo protocol; Sample #16 from HEK luc transfections (2-day/ mock)
qRT-PCR
> Plate 23
--> 750 nM Primers (& cDNA dilution)
- mCh1 (1:1000)
- GAPDH 21A (1:1000)
- ACTC1 35A (1:10)
- ATOH1 46D "
- CASP10 41A "
- CDKN2A 7C "
- EOMES 42A "
- GRHL2 37C "
> Templates, use 2 μL
- KAH157-1, 4/08/11 A
- KAH157-1 +dox, 4/08/11 A
- KAH157-1, 4/08/11 B
- KAH157-1 +dox, 4/08/11 B
> Plate 24
--> 750 nM Primers (& cDNA dilution)
- MMP12 31B (1:10)
- NPPA 40A "
- OTX2 43D "
- SST 45C "
- THPO 33A "
- TNF 34A "
> Templates, use 2 μL
- KAH157-1, 4/08/11 A
- KAH157-1 +dox, 4/08/11 A
- KAH157-1, 4/08/11 B
- KAH157-1 +dox, 4/08/11 B
| Reagent |
1 rxn |
Primer mixes 1-6 (x13)
|
| diluted cDNA |
2.0 |
---
|
| SYBR Green mix |
7.5 |
97.5
|
| 750 nM primers |
3.0 |
39.0
|
| dH2O |
2.5 |
32.5
|
| |
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
RNA prep/ cDNA
> Sample "#16 2-day/mock"; frozen in TRIzol (-80°C)
> Use TRIzol protocol up to chloroform extraction
> Apply aqueous phase to RNeasy column (suggestion from Mara) and follow rest of RNeasy protocol
> RNA prep: A260 = 1.393, 260/280 = 1.83, 55.7 ng/μL
> Yield not very high, but peak looks good, decent quality RNA
> cDNA synthesis: Superscript III kit; use 8 μL RNA (max)
> Will use this cDNA to test luc primers when they arrive
|
Project name
Main project page
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