RNA Total Tumbuhan Kompleks (plus) Kit Ekstraksi Jumlah Kecil

1. Komponen kit reagen

2. Penyimpanan

This reagent kit can be stored at room temperature (15-25℃) in a dry environment and is stable for 12 bulan.

3. Petunjuk Penggunaan Kit Reagen

3.1 Kit ini ditujukan untuk tujuan penelitian biologi molekuler dan tidak boleh digunakan untuk diagnosis atau pengobatan penyakit.

3.2 Beberapa komponen dalam kit mengandung bahan pengiritasi; disarankan untuk mengambil tindakan pencegahan yang diperlukan (seperti memakai pakaian pelindung dan kacamata).

3.3 Penggunaan kit ini memerlukan peralatan tambahan seperti centrifuge berkecepatan tinggi, mandi air (mandi logam), pencampur pusaran, etanol anhidrat, nitrogen cair, khloroform, air deionisasi steril, dan tabung EP.

4. Pengantar Kit Reagen

This RNA purification reagent kit provides a fast and effective purification of plant total RNA containing polysaccharides, lipids, polyphenols, and other components. It is suitable for most complex plant tissues. In general, lipid-rich plant tissues contain a high content of lipid compounds, which significantly affect RNA extraction efficiency. This kit utilizes exclusive inhibitor removal columns to effectively eliminate lipids from plant samples, as well as to remove DNA contamination. If the experiment is sensitive to DNA, it is recommended to use intron-spanning primers for downstream experiments.

The RNA rapid purification reagent kit can extract plant total RNA (including nuclear RNA and cytoplasmic RNA) di dalam 1 jam. The extracted RNA can be directly used for RT-PCR, Northern blotting, dll.. The entire purification process does not require toxic reagents such as chloroform, making the RNA purification reagent kit suitable for various other samples.

5. Prinsip dan Prosedur Eksperimental

6. Proses Ekstraksi

Tindakan pencegahan sebelum memulai percobaan:

A. Mencuci Penyangga 1: Prior to use, add the specified amount of absolute ethanol as indicated on the reagent bottle label. Check the label to confirm the addition of absolute ethanol.

B. Buffer Elusi adalah a 0.1x solusi TE with a minimal amount of EDTA. If EDTA may affect subsequent experiments, it is recommended to replace the elution buffer with sterile deionized water.

C. RNA Extraction Buffer I contains a small amount of phenol, which may precipitate. Sebelum digunakan, mix thoroughly by warming in a water bath; after use, store away from light.

1. Pemrosesan Sampel:

A. Pengumpulan dan Penyimpanan Bahan:

Jika bahan yang baru dikumpulkan tidak dapat langsung digunakan, place them in liquid nitrogen and store them at -80℃.

B. Whenever possible, collect fresh materials as they contain fewer polysaccharides and polyphenols.

2. Grind approximately 100 mg of fresh samples or not more than 20 mg of dry material with liquid nitrogen.

3. Menambahkan 600μl of RNA Extraction Buffer I, ensuring there are no tissue clumps in the ground sample. Tissue clumps are difficult to lyse and can reduce RNA yield.

4. Vortex for 30 S.

5. Transfer the lysate to aninhibitor removal purification column, sentrifugasi di 12,000 rpm untuk 5 menit.

(Catatan: Lipid-rich plant materials may contain many lipid compounds at this step, which can affect RNA extraction. Remove these substances during this step. If buffer residue remains in the inhibitor removal purification column during centrifugation, extend centrifugation time appropriately.)

6. Transfer the obtained supernatant to a DNA removal column, sentrifugasi di 12,000 rpm for 30s, and collect the filtrate (Catatan: RNA is present in the filtrate).
7. Menambahkan 250μl etanol absolut, campur dengan cara dipipet. If there is a small amount of precipitation, it does not affect subsequent experiments. Transfer the liquid to an RNA purification column, sentrifugasi di 12,000 rpm for 30s, discard the flow-through.
8. Menambahkan 700μl of inhibitor removal buffer, sentrifugasi di 12,000 rpm for 30s, discard the flow-through.
9. Menambahkan 700μl dari washbuffer 1 to the RNA purification column, sentrifugasi di 12,000 rpm for 30s, discard the flow-through.
10. Menambahkan 500μl dari wash buffer 1 to the RNA purification column, sentrifugasi di 12,000 rpm untuk 3 menit, discard the flow-through.
11. Place the RNA purification column into a new 1.5ml centrifuge tube, air-dry the membrane at room temperature for 2 menit.

(Catatan: Confirm that absolute ethanol has been added to wash buffer 1. The presence of ethanol has a serious impact on subsequent experiments, so drying the membrane is crucial. Setelah sentrifugasi, ensure no ethanol is present before elution, then discard the waste and collection tube. After washing with wash buffer 1, the membrane on the RNA purification column should have only a slight color. Setelah sentrifugasi, carefully remove the RNA purification column, ensuring it does not touch the collection tube to avoid ethanol interference.)

12. Aerially pipette 50-100μl buffer elusi ke membran, sentrifugasi di 12,000 rpm untuk 1 menit, and collect the RNA solution.

(Catatan: Eluting RNA with 50 μl of elution buffer can increase RNA concentration but reduces total RNA yield.)