Which protocol of DNA extraction from plant oil-containing seeds take fast and good result in purity that can use in RT-PCR(Real Time polymerase chain reaction)?

  • $\begingroup$ I guess you mean realtime with RT, right? $\endgroup$
    – Chris
    Sep 10 '14 at 15:17
  • $\begingroup$ @Chris yes that's right $\endgroup$
    – M007
    Sep 10 '14 at 15:21
  • $\begingroup$ Maybe this is interesting for you. $\endgroup$
    – Chris
    Sep 10 '14 at 15:54
  • $\begingroup$ @Chris you should post that as answer. You can copy paste the protocol $\endgroup$
    Jan 14 '15 at 8:34

The paper "Modified method for combined DNA and RNA isolation from peanut and other oil seeds." has a nice protocol for isolating DNA and RNA (which later can either be differentially digested or precipitated) from oil seeds for PCR. You can find the complete paper in the link above, the materials and methods sections says the following for the extraction method:

RNA and DNA isolation

Approximately 1–1.2 g of dried seed of each genotype was utilized in extraction. Seeds were frozen with liquid nitrogen and pulverized by grinding with mortar and pestle until a fine powder. Tissue was placed into 10 mL solution I (100 mM Tris, 150 mM LiCl, 50 mM EDTA, 1.5 % SDS, 2 % PVP-40, 1.5 % b-mercaptoethanol) buffer, pH 7.5, and mixed thoroughly by vortexing or inversion.

To the homogenized tissue, a 5 mL acid phenol, pH 4.3 (Fisher Scientific, Pittsburg, PA) was added and tissue was mixed thorough for 5 min, followed by the addition of 5 mL chloroform:isoamyl alcohol (24:1) and mixed for an additional 5 min. The homogenate was centrifuged for 10 min and the upper aqueous layer (around 9 mL) was transferred to a fresh 40 mL tube containing 9 mL of solution II (2 M guanidinium thiocyanate, 0.5 M sodium citrate, 1.5 M ammonium acetate, 1 % N-lauroylsarcosine) buffer, pH 5.2, mixed by gentle inversion, and allowed to incubate for 10 min at room temperature.

After incubation, a 4.5 mL phenol:chloroform:isoamyl alcohol (25:24:1), pH 6.7 was added, mixed thoroughly for 5 min, centrifuged for 10 min at 13,000g, and the aqueous layer (around 16 mL) was transferred to a new 40 mL tube.

To the transferred solution, 9 mL 1.2 M NaCl and 9 mL isopropanol were added, mixed, placed on ice for at least 1 h, and centrifuged for 15 min at 13,000g. The liquid was discarded and the resulting pellet was washed with 2 mL cold 70 % ethanol, dried, and resuspended in 4 mL RNase-free water.

4 mL 4 M LiCl was added, mixed, placed on ice in the refrigerator for at least 1 h, and centrifuged for 15 min at 4°C at 13,000g. The supernatant (around 8 mL) was transferred to a new 40 mL tube for DNA isolation and the pellet was retained for RNA isolation. For RNA isolation, the pellet was washed with 2 mL ice-cold 70 % ethanol, dried, and resuspended in 750 lL RNase-free water.

For DNA isolation, 8 mL isopropanol was added to the transferred supernatant tube, mixed well, centrifuged for 5 min, discarded the liquid, washed pellet with 2 mL ice-cold 70 % ethanol, dried, and resulting pellet was resuspended in 500 lL 10 mMTris, pH7.5. The amount of DNA or RNA was determined using Nanodrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE).


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