Tissue DNA/RNA Extraction Kit

1. Komponen kit reagen

2. Penyimpanan

Kit reagen ini harus disimpan pada suhu kamar (15-25℃) in a dry condition and is stable for 12 bulan. Proteinase K contains a stabilizer, allowing it to be transported at room temperature; Namun, 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

The sample DNA/RNA simultaneous isolation and purification kit provide a fast and efficient purification solution for tissue sample DNA/RNA. This kit offers two sets of lysis systems, suitable for various sample tissues and cells, including most plants, binatang, bakteri, dll..

The DNA/RNA rapid purification kit can extract total DNA/RNA from samples within 1 jam. The extracted DNA/RNA can be directly used for RT-PCR, Northern blotting, dll.. Seluruh proses pemurnian tidak memerlukan reagen beracun seperti fenol-kloroform.

5. Prinsip dan Prosedur Eksperimental

6. Proses Ekstraksi

Tindakan pencegahan sebelum memulai percobaan:

A. Sebelum digunakan, Tambahkan jumlah etanol absolut yang ditentukan MencuciPenyangga 1 Menurut label pada botol reagen, and mark a check on the label to indicate the addition of absolute ethanol.

B. Buffer Elusi adalah a 0.1x solusi TE with a minimal amount of EDTA. If EDTA has an impact on subsequent experiments, it is recommended to use sterile deionized water as a substitute for the elution buffer.

C. RNA Extraction Buffer I is designed for the extraction of plant and fungal samples, while RNA Extraction Buffer IV is primarily used for animal tissues, darah, and other sample tissues.

1. Pemrosesan Sampel:
2. Plant/animal tissue materials: Collect samples using liquid nitrogen, grind the collected material directly in liquid nitrogen, and proceed to step 2 (recommended to use RNA Extraction Buffer I).
3. Cell tissues: Centrifuge and collect <107 suspended cells in a 1.5 ml tabung sentrifugasi, quickly proceed to step 2 (recommended to use RNA Extraction Buffer III).
4. Menambahkan 500μl RNA Extraction Buffer I/RNA Extraction Buffer IV, 10μl Proteinase K, vortex and mix thoroughly. Ensure gentle mixing in this step to prevent mechanical cutting of tissue DNA and reduce DNA yield.
5. Transfer the lysate to a pemurnian DNA kolom, sentrifugasi di 13,000 rpm untuk 2 menit, collect the filtrate (RNA is present in the filtrate). Pada titik ini, DNA is adsorbed on the DNA column and can be briefly stored at 4°C while simultaneously collecting RNA in the next steps.

(Catatan: If the sample is plant tissue, sentrifugasi di 13,000 rpm untuk 10 menit, and add the supernatant to the DNA binding column.)

4. Precisely estimate the volume of the filtrate, menambahkan 5 times thevolume of absolute ethanol, aduk rata. Jika terjadi curah hujan, it does not affect subsequent experiments.
5. Add the obtained liquid to the RNA extraction purification column (sekitar 650-700μl setiap kali), centrifuge di atas 8,000 rpm untuk 1 menit, Buang cairan limbah yang dikumpulkan, dan masukkan kembali tabung pengumpul ke dalam kolom pemurnian untuk langkah selanjutnya.
6. Ulangi langkah 5, add the remaining liquid to the RNA extraction purification column, centrifuge di atas 8,000 rpm untuk 1 menit, discard the waste liquid and collection tube, place the RNA extraction purification column in a new collection tube, and prepare for simultaneous collection of DNA.
7. Menambahkan 600buffer penghapusan penghambat μl to the DNA extraction purification column and RNA extraction purification column, centrifuge di atas 8,000 rpm untuk 1 menit, Buang cairan limbah, and reinsert the DNA/RNA purification column into the collection tube for the next step.
8. Menambahkan 700μl buffer cuci 1 to the DNA extraction purification column and RNA extraction purification column, sentrifugasi di 14,000 rpm (20,000×g) untuk 2 menit. Extend centrifugation time appropriately to ensure the membrane is thoroughly dried.

(Catatan: Konfirmasikan bahwa etanol absolut telah ditambahkan ke Wash Buffer 1. The presence of ethanol has a significant impact on subsequent experiments, so membrane drying is crucial. Setelah sentrifugasi, ensure no ethanol is present before elution, then discard the waste liquid and collection tube.

After washing with Wash Buffer 1, the membrane of the RNA purification column should only have a slight color. Setelah sentrifugasi, Lepaskan kolom pemurnian RNA dengan hati -hati, ensuring no contact with the collection tube to avoid ethanol interference.)

9. Place the DNA/RNA purification column into a new centrifuge tube, menetes 50-100 μl Elution Buffer onto the membrane, Inkubasi pada suhu kamar untuk 5 menit (15°C-25°C), centrifuge di atas 8,000 rpm untuk 1 menit.

(Catatan: Eluting nucleic acids with 50 μl Elution Buffer can increase the nucleic acid concentration but decrease the total nucleic acid yield.)

• Catatan:

1. Umumnya, larger elution volumes result in higher elution efficiency, but to increase nucleic acid concentration, the elution volume can be reduced, but not below 50 μl.
2. RNA, in the presence of RNA Extraction Buffer I/RNA Extraction Buffer III, is not degraded by RNAse but should be distinguished from DNA in subsequent experiments. Use RNAse-free consumables for RNA separation whenever possible.