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

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03/12/11

  • ✓ 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
Reagent 1 rxn Primer mix (5x) Template mix (25x)
cDNA 0.5 --- 12.5
10 μM primers 1.0 5.0 ---
2x GoTaq Green 10.0 50.0 ---
dH2O 8.5 --- 212.5
  20.0 μL

--> 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/ ∞


File:KAH 031211 gel1.tif

--> 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