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

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03/17/11

 * &#x2713; qRT-PCR: Pc-TF -/+ dox, plate 5, 6
 * &#x2713; 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

--> 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

--> 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/ ∞



--> 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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