User:Karmella Haynes/Notebook/Polycomb project/2011/04/10

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04/10/11

  • ✓ RT-PCR: test primers on 129-4



RT-PCR
> Test failed RT-PCR primers on KAH129-4 (36 rxns total)
> Primers:
1-4. FGF10 36A-D (114, 117, 119, 80 bp)
5-8. INS 38A-D (106, 110, 107, 109 bp)
9-12. MYF5 39A-D (113, 113, 81, 81 bp)
13-16. HHEX 47A-D (86, 80, 87, 113 bp)
17-20. IL4 48A-D (97, 102, 106, 94 bp)
21-24. SLC6A4 49A-D (84, 86, 80, 83 bp)
25-28. NFE2 52A-D (90, 88, 118, 120 bp) (Accidentally omitted NFE2 A, B)
29-32. NEUROG1 53A-D (105, 117, 92, 98 bp)
33-36. SOX1 54A-D (84, 80, 80, 102 bp)


> Template:

  1. KAH129-4 +dox (4/08/11) 1:10


Reagent 1 rxn Template mix (38x)
cDNA 0.5 19.0
10 μM primers 1.0 ---
2x GoTaq Green 10.0 380.0
dH2O 8.5 323.0
  20.0 μL

--> 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/ ∞


File:KAH 041011 gel1.tif

--> Conclusions:

  • None worked. Discontinue testing these primers.