Quint Lab:arabidopsis rna isolation protocols (including siliques)

From OpenWetWare

Revision as of 09:35, 21 January 2010 by Nadine Schumann (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Arabidopsis RNA isolation (including isolation from siliques)

(L.Oñate-Sánchez,J.Vicente-Carbajosa: DNA-free RNA isolation protocols for Arabidopsis thaliana,including seeds and siliques,BMC Research Notes 2008, 1:93)

Note:

PROTOCOL 1 (vegetative tissues)

  1. Add 1200 µl of cell lysis solution (2% SDS, 68 mM sodium citrate, 132 mM citric acid, 1 mM EDTA) to ground tissue and homogenize quickly by vortexing 2 s and inverting and flicking the tube gently. Leave tubes at room temperature up to 5 min.
  2. Add 400 µl of protein-DNA precipitation solution (4 M NaCl , 16 mM sodium citrate, 32 mM citric acid) to the cell lysate, mix inverting the tubes gently and incubate at 4°C for at least 10 min. Spin at 4°C for 10 min.
  3. Transfer supernatant (s/n) to a new tube, add 1000 µl isopropanol and mix the sample by inverting gently the tube. Spin 4 min and carefully pour off the s/n. Wash pellet with 70% ethanol, air-dry RNA and resuspend in 100 µl DEPC- water.
  4. Add 80 µl Dnase I-Lösung (10 µl gelöste DNase + 70 µl Puffer RDD [QIAGEN RNase-Free DNase Set for on-column digestion]) Incubate 15 min at RT.
  5. Add 280 µl DEPC-water to the 120 µl of RNA, 100 µl of NH4Ac 7.5 M (sodium acetate can also be used) and 1600 µl 100% ethanol and mix well. Spin 20 min at 4°C, wash pellet with 70% ethanol, air-dry RNA and resuspend in 40 µl DEPC-water.

PROTOCOL 2 (seeds and siliques)

  1. Transfer ground tissue to a cooled 15 ml-falcon tube and quickly add 2200 µl of extraction buffer (0.4 M LiCl, 0.2 M Tris pH:8, 25 mM EDTA, 1% SDS) and 2200 µl chloroform. Vortex 10 s and keep on ice until all samples are ready. Spin for 3 min.
  2. Transfer supernatant (s/n) to a new tube, add 2000 µl of water-saturated acidic phenol, vortex thoroughly, add 800 µl of chloroform and spin 3 min.
  3. Transfer s/n to a new tube, add 1/3 volume of 8 M LiCl and mix. Precipitate at -20 °C for 1 hour (overnight at 4°C is also possible) and spin for 30 min at 4°C
  4. Dissolve pellet in 100 µl DEPC-water. Add 80 µl Dnase I-Lösung (10 µl DNase + 70 µl Buffer RDD [QIAGEN RNase-Free DNase Set for on-column digestion]). Incubate 15 min at RT.
  5. Add 1880 µl DEPC-water, 28 µl 3 M NaAc pH 5.0 and 1000 µl ethanol, mix well and spin 10 min at 4°C to precipitate carbohydrates.
  6. Transfer s/n to a new tube, add 172 µl 3 M NaAc pH 5.0 and 3000 µl ethanol, mix well and leave at –20°C for at least 1 hour. Spin 20 min at 4°C, wash pellet with 70% ethanol, air-dry RNA and resuspend in 40 µl DEPC- water.
Personal tools