User:Marcel Quint/Notebook/medicago/2008/04/25

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material for RNA isolation, cDNA and RT-PCR

  • harvest ~50 mg of leaves, petioles, flowers, stem, roots from 58d.o. medicago truncatula A17 plant (4 biological repeats each) → freeze in liquid nitrogen
  • RNA isolation following Qiagen protocol; quantities:

petiole 1: 352 ng/μL
petiole 2: 446 ng/μL
petiole 3: 1703 ng/μL
petiole 4: 402 ng/μL

stem 1: 154 ng/μL
stem 2: 834 ng/μL
stem 3: 418 ng/μL

root 1: 405 ng/μL
root 2: 540 ng/μL
root 3: 468 ng/μL
root 4: 693 ng/μL

leaves 1: 2335 ng/μL
leaves 2: 2116 ng/μL
leaves 3: 1326 ng/μL
leaves 4: 291 ng/μL

flowers 1: 2591 ng/μL
→ store in -80°C

  • cDNA synthesis using supersript III following manufacturer's protocol; at first only one sample of each tissue → use 2 µg RNA of petiole 2, stem 2, root 4, leaves 2, flowers 1

→ store in -20°C


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