User:Karmella Haynes/Notebook/Polycomb project/2011/03/12

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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03/12/11

 * &#x2713; PCR: test new RT-PCR primers on KAH126-1

PCR > Test new RT-PCR primers on KAH126-1 (96 rxns total) > Primers: 1-4. ACTC1 35A-D (80, 80, 80, 80 bp) 5-8. FGF10 36A-D (114, 117, 119, 80 bp) 9-12. GRHL2 37A-D (116, 117, 84, 105 bp) 13-16. INS 38A-D (106, 110, 107, 109 bp) 17-20. MYF5 39A-D (113, 113, 81, 81 bp) 21-24. NPPA 40A-D (115, 90, 114, 104 bp) > Templates:
 * 1) KAH126-1 -dox 1:1
 * 2) KAH126-1 -dox 1:10
 * 3) KAH126-1 +dox 1:1
 * 4) KAH126-1 +dox 1:10

--> 4 template mixes: aliquot 9.0 μL --> 24 primer mixes: aliquot 11.0 μL

--> 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) ACTC1: all worked, use 35A
 * 2) FGF10: 36C shows band, but shorter than predicted size
 * 3) GRHL2: use 37C
 * 4) INS: failed
 * 5) MYF55: failed
 * 6) NPPA: use 40A

Next: optimize qPCR; may be able to use less DNA


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