Super-Pure RNA Extraction Kit (TRIzol)

1. Komponen kit reagen

2. Penyimpanan

Kit reagen ini harus disimpan pada suhu kamar (15-25℃) in a dry environment and is stable for 12 bulan. DNase I contains a preservative, memungkinkan transportasi pada suhu kamar, tapi untuk penyimpanan jangka panjang, itu harus dijaga pada -20℃.

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 kit utilizes the traditional TRIzol combined with column membrane method for the rapid purification of plant, animal, jaringan, cell, microbial RNA, and fungal hyphae RNA. It is suitable for most species. This RNA purification kit can be applied to plant tissues exceeding 100 mg. The RNA extracted with this kit has extremely low DNA content. If sensitivity to DNA in experiments is a concern, it is recommended to use DNase I digestion on the column.

The RNA Fast Purification Kit can extract total RNA from samples (including nuclear RNA and cytoplasmic RNA) di dalam 1 jam. The extracted RNA can be directly used for RT-PCR, Northern blotting, dan aplikasi lainnya.

5. Prinsip dan Prosedur Eksperimental

6. Proses Ekstraksi

Tindakan pencegahan sebelum memulai percobaan:

A. Sebelum digunakan, add the specified amount of absolute ethanol to MencuciPenyangga 1 according to the label on the reagent bottle, and check the box on the label to indicate that absolute ethanol has been added.

B. Buffer Elusi adalah a 0.1x solusi TE mengandung minimal EDTA. Jika EDTA mempengaruhi percobaan selanjutnya, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Pemrosesan Sampel:

A. Jaringan: If fresh materials cannot be used immediately, place them in liquid nitrogen and store them at -80°C. Bahan kering dapat disimpan pada suhu kamar. Grind 30~80 mg of tissue in liquid nitrogen and add 1 ml Trizol buffer for homogenization.

B. Monolayer Cultured Cells: Aspirate the culture medium and add 1 ml Trizol buffer for homogenization.

C. Cell Suspension: Centrifuge to collect cells and add 1 ml Trizol buffer for homogenization.

2. After adding Trizol buffer to the sample, mix thoroughly by pipetting, allow complete sample lysis, dan inkubasi pada suhu kamar selama 5 menit.

3. Add chloroform in a ratio of 200 μl per 1 ml of Trizol buffer, vigorously shake for 30 detik, dan inkubasi pada suhu kamar selama 2 menit.

4. Centrifuge the lysate at 4°C, 12,000 rpm untuk 10 menit. RNA will be present in the upper aqueous phase.

5. Pindahkan supernatan dengan hati-hati ke dalam tabung sentrifugasi baru, avoiding the collection of the middle and bottom layers. (Catatan: Sekitar 400 μl cairan dapat ditransfer, which may be less for some species.)

6. Add an equal volume of pre-chilled absolute ethanol, mix quickly. (Misalnya, menambahkan 400 μl lisat, menambahkan 400 μl etanol absolut. Jika volume lisat kurang dari 400 μl, reduce the amount of absolute ethanol proportionally. A slight precipitate may form after adding ethanol, but it does not affect subsequent experiments.)

7. Transfer the obtained liquid to an RNA purification column (sekitar 650-700 μl per time), centrifuge lebih dari 8,000 rpm untuk 1 menit, membuang sampah yang dikumpulkan, dan masukkan kembali tabung pengumpul ke dalam kolom pemurnian untuk langkah selanjutnya.

8. Ulangi langkah 7, adding the remaining liquid to the RNA purification column, centrifuge lebih dari 8,000 rpm untuk 1 menit, discard the waste and the collection tube.

9. Place the RNA purification column in a new collection tube, menambahkan 300 μl of Inhibitor Removal Buffer, centrifuge lebih dari 8,000 rpm untuk 1 menit, membuang limbahnya, and reinsert the RNA purification column into the tube for the next step.

10. Menambahkan 80 μl of DNase Iworking solution to the RNA purification column, incubate at 20°C to 30°C for 15 menit. (Prepare DNase I working solution: 62 μl RNase-Free Water, 8 μl 10× Reaction Buffer, Dan 10 μl DNase I, make up to 80 μl DNase I working solution.)

11. Menambahkan 300 μl of Inhibitor Removal Bufferto the RNA purification column, centrifuge lebih dari 8,000 rpm untuk 1 menit, membuang limbahnya, and reinsert the RNA purification column into the tube for the next step.

12. Menambahkan 700 μl Buffer Cuci 1to the RNA purification column, sentrifugasi di 14,000 rpm (20,000×g) untuk 2 menit, extend the centrifugation time if needed for a drier membrane. (Catatan: Confirm the addition of ethanol to Wash Buffer 1; ethanol presence significantly affects subsequent experiments. Ensure the membrane is dry after centrifugation before elution. Buang sampah dan tabung pengumpul. After using Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. Carefully remove the RNA purification column after centrifugation, ensuring it does not touch the collection tube to avoid ethanol contamination.)

13. Place the RNA purification column in a new centrifuge tube, drip 100 μl Buffer Elusike membran, incubate at room temperature for 5 menit (15°C to 25°C), and centrifuge at more than 8,000 rpm untuk 1 menit. (Catatan: Eluting RNA with 50 μl of Elution Buffer can increase RNA concentration but decrease total RNA yield.)

14. Repeat the previous step. (Catatan: A new centrifuge tube can be used to collect the RNA eluted the second time or continue using the original collection tube to collect RNA.)