Kit Ekstraksi DNA Genom Bakteri

• Komponen kit reagen

• Penyimpanan

Kit reagen ini harus disimpan pada suhu kamar (15-25℃) dan dalam kondisi kering, dengan umur simpan 12 bulan. Kolom pemurnian ekstraksi DNA dapat disimpan 1 tahun di lingkungan sejuk dan kering. Lysozyme, Proteinase K and RNase A contain preservatives, memungkinkan transportasi pada suhu kamar, tapi untuk penyimpanan jangka panjang, mereka harus disimpan pada -20℃.

• Petunjuk Penggunaan Kit Reagen

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

3.2 Beberapa komponen dalam kit reagen mengandung bahan iritan. Tindakan perlindungan seperti mengenakan pakaian pelindung dan kacamata direkomendasikan.

3.3 Selama penggunaan kit reagen ini, mesin sentrifugal berkecepatan tinggi, mandi air (mandi logam), pencampur pusaran, etanol anhidrat, air deionisasi steril, dan tabung EP perlu disiapkan oleh pengguna.

• Pengantar Kit Reagen

This kit provides a rapid and effective purification method for isolating DNA from various bodily fluids and bacterial culture fluids. It utilizes a silicon-based purification column that selectively adsorbs nucleic acids. With specific buffer solutions, bacterial DNA samples can be extracted within 30 menit. The entire purification process does not require toxic reagents such as phenol-chloroform. The extracted DNA can be directly used for downstream experiments like PCR, Blot Selatan, dan lain-lain.

• Prinsip dan Prosedur Eksperimental

• Proses Ekstraksi

Sebelum Memulai Eksperimen:

1. Reagent Buffer B and C dapat mengendap pada kondisi suhu rendah. We recommend heating at 65℃for 5 menit. After the precipitate dissolves, it can be used normally.
2. Sebelum digunakan, add the specified amount of anhydrous ethanol to Cuci Buffer 1as indicated on the bottle label. Mark a check on the label to indicate the addition of anhydrous ethanol.
3. Buffer Elusi adalah a0.1x solusi TEcontaining minimal amounts of EDTA. Jika EDTA mungkin mempengaruhi percobaan selanjutnya, it is advisable to substitute Elution Buffer with sterile deionized water.
4. Penanganan Sampel:
5. Take around 1 ml of bacterial culture, sentrifugasi di 12,000 rpm untuk 1 menit, aspirate the supernatant as much as possible, menambahkan 200μl Reagen Buffer Bto the bacterial cell solution. Umumnya, residual RNA has minimal impact on downstream experiments. If RNA interference needs to be eliminated, menambahkan 10μl of RNaseA (10mg/ml) to the mixture, incubate at 37°C for 2 minutes with vortexing during the period.
6. If the sample being processed contains Gram-positive bacteria, menambahkan 10μl of Lysozyme (10 mg/ml)Dan 200μl Reagen Buffer B. Umumnya, residual RNA has minimal impact on downstream experiments. If RNA interference needs to be eliminated, menambahkan 10μl of RNaseA (10mg/ml) to the mixture, incubate at 37°C for 15-30 minutes with vortexing during the period.
7. Menambahkan 10μl of Proteinase K (10 mg/ml), thoroughly invert and mix, digest at 65°C for 2 menit. Selama periode ini, invert and mix the sample solution 6-7 times until the sample solution becomes clear after digestion.
8. Menambahkan 200μl Reagen Buffer Cto the lysate and mix. Jika muncul endapan putih, it can be left to settle; it won’t affect subsequent experiments once the precipitate disappears.
9. Menambahkan 200μl of ethanol, aduk rata. Some precipitation might occur but won’t affect subsequent experiments.
10. Transfer the obtained liquid into a DNA extraction purification column, leave at room temperature for 2 menit, sentrifugasi di 12,000 rpm untuk 30 detik. Discard the collected waste and reinsert the collection tube into the purification column for the next step.
11. Menambahkan 600μl Buffer Cuci 1, sentrifugasi di 12,000 rpm untuk 30 detik, membuang limbahnya, and reinsert the DNA extraction purification column into the holder.

(Catatan: Ensure ethanol has been added to Wash Buffer 1.)

7. Menambahkan 500μl Buffer Cuci 1 ke kolom pemurnian ekstraksi DNA, sentrifugasi di 12,000 rpm untuk 2 menit, extending the centrifugation time as needed for a drier membrane.
8. Tempatkan kolom pemurnian ekstraksi DNA (holder) ke dalam tabung centrifuge yang baru, membuka, dan panaskan pada suhu 65°C selama 2 menit. Extend this step as necessary to evaporate ethanol, preventing ethanol residues from affecting downstream experiments.
9. Menambahkan 50-100μl Buffer Elusike membran kolom, sentrifugasi di 12,000 rpm untuk 2 menit.

(Catatan: 1. Mengelusi DNA dengan 50 μl of Elution Buffer can increase DNA concentration but reduce the total DNA yield. 2. The eluted DNA from the eluate can be reapplied to the DNA extraction purification column, centrifuge again at 12,000 rpm untuk 2 minutes to enhance DNA yield.)