User:Karmella Haynes/Notebook/Polycomb project/2010/09/23

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09/23/10

 * &#x2713; RT-PCR: Repeat KAH154-2, 156-5, correct loading

RT-PCR > Pc-ATF NS repeats; balance loading for KAH154-2 and 156-5 samples (see 8/26/10 for GAPDH RT-PCR) --> Templates --> Primers
 * 1) KAH154-2 -dox (0.5x)
 * 2) KAH154-2 +dox (4x)
 * 3) KAH156-5 -dox (0.5x)
 * 4) KAH156-5 +dox (2x)
 * 1) p16INK 7C (94 bp) (4 rxns)
 * 2) MMP12 31A (127 bp) (4 rxns)
 * 3) THPO 33A (103 bp)  (4 rxns)
 * 4) TNF 34A (119 bp) (4 rxns)
 * 5) GAPDH 21A (100 bp), 1:1000 cDNA (4 rxns)
 * 6) mCh (100 bp), 1:500 cDNA (4 rxns)

--> Use 0.5 2x diluted cDNA + 1.5 dH2O for KAH154-2 -dox --> Use 2.0 cDNA for KAH154-2 +dox --> Use 0.5 2x diluted cDNA + 1.5 dH2O for KAH156-5 -dox --> Use 1.0 cDNA + 1.0 dH2O for KAH156-5 +dox

--> PCR (96-well)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
 * 72°C/ 3 min.
 * 4°C/ ∞



> Conclusions:
 * p16, GAPDH, and mCh are good
 * Forgot to use 1:10 dilution for MMP12, THPO, and TNF; repeat using the same adjustments as above, but use the 1:10 dilutions as starting cDNA stocks




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