Bacterial Total RNA (più) Extraction Kit

1. Componenti del kit di reagenti

Specifiche	50T	100T
Gatto. NO.	SN0321-D	SN0322-D
RNA Extraction Columns (impostato)	50（套）	100（套）
DNA Clean-Up Columns (impostato)	50（套）	100（套）
RNA Extraction Buffer I	30ml	2× 30 ml
Inhibitor Removal Buffer	30ml	2×30 ml
Tampone di lavaggio 1	15 ml	2×15 ml
Tampone di eluizione	20ml	2×20 ml
Lisozima	5ml	2x5ml
Manuale di istruzioni	1	1

2. Magazzinaggio

Questo kit di reagenti deve essere conservato a temperatura ambiente (15-25℃) in a dry environment and can be preserved for 12 mesi. Lysozyme contains a preservative, allowing for transportation at room temperature; Tuttavia, for long-term storage, it should be stored at -20℃

3. Istruzioni per l'uso del kit di reagenti

3.1 Questo kit è destinato a scopi di ricerca in biologia molecolare e non deve essere utilizzato per la diagnosi o il trattamento di malattie.

3.2 Alcuni componenti del kit contengono sostanze irritanti; è opportuno prendere le dovute precauzioni (come indossare indumenti protettivi e occhiali protettivi).

3.3 L'utilizzo di questo kit richiede apparecchiature aggiuntive come una centrifuga ad alta velocità, bagnomaria (bagno di metallo), miscelatore a vortice, etanolo anidro, azoto liquido, cloroformio, acqua deionizzata sterile, e tubi EP.

4. Introduzione al kit di reagenti

This RNA purification kit provides a rapid and efficient purification solution for bacterial RNA, suitable for most bacterial tissues. The kit employs DNA removal technology to effectively eliminate genomic DNA, and the resulting RNA samples typically do not require on-column digestion, reducing the risk of RNA degradation.

The RNA Fast Purification Kit allows for the extraction of total bacterial RNA (including nuclear RNA and cytoplasmic RNA) entro 1 ora. The extracted RNA can be directly used for RT-PCR, Northern blotting, eccetera. The entire purification process does not involve toxic reagents such as phenol-chloroform, making the RNA purification kit well-suited for various sample types.

5. Principi e procedure sperimentali

6. Processo di estrazione

Precauzioni prima di iniziare l'esperimento:

UN. Prima dell'uso, add the specified amount of absolute ethanol to Tampone di lavaggio 1 as indicated on the reagent bottle label, and mark with a check to indicate the addition of absolute ethanol.

B. Il tampone di eluizione è un 0.1x soluzione TE contenente una quantità minima di EDTA. If EDTA may impact subsequent experiments, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Elaborazione del campione:

UN. Batteri gram-positivi: Centrifuge the bacterial suspension at 4℃, 12,000 giri al minuto per 2 minuti, collect bacterial cells (1.53 ml), scartare il surnatante, aggiungere 100μl Lysozyme (10mg/ml), thoroughly mix the bacterial cells, e incubare a temperatura ambiente per 15-30 minuti.

B. Batteri gram-negativi: Centrifuge the bacterial suspension at 4℃, 12,000 giri al minuto per 2 minuti, collect bacterial cells (1.53 ml), scartare il surnatante, aggiungere 10μl Lysozyme (10mg/ml), thoroughly mix the bacterial cells, e incubare a temperatura ambiente per 3-5 minuti.

2.Aggiungere 400μl RNA Extraction Buffer I, vortex to mix thoroughly.

3. Transfer the lysate to a DNA clearance purification column, centrifugare a 13,000 giri al minuto per 2 minuti, collect the filtrate (RNA is present in the filtrate).

4. Accurately estimate the volume of the filtrate, aggiungere 0.5 times the volume of absolute ethanol, mescolare accuratamente; if precipitation occurs, it does not affect subsequent experiments.

5. Add the obtained liquid to the RNA extraction purification column (circa 650~700μl ogni volta), centrifuge at more than 8,000 giri al minuto per 1 minuto, smaltire il liquido di scarto raccolto, and reinsert the collection tube into the RNA extraction purification column for the next step.

6. Ripeti il ​​passaggio 5, add the remaining liquid to the RNA extraction purification column, centrifuge at more than 8,000 giri al minuto per 1 minuto, eliminare il liquido di scarto e il tubo di raccolta.

7. Place the RNA extraction purification column into a new collection tube, aggiungere 600μl Inhibitor Removal Buffer, centrifuge at more than 8,000 giri al minuto per 1 minuto, eliminare il liquido di scarto, and reinsert the RNA extraction purification column into the tube for the next step.

8. Aggiungere 700μl Wash Buffer 1 to the RNA extraction purification column, centrifugare a 14,000 giri/min (20,000× g) per 2 minuti, extend the centrifugation time appropriately to ensure the membrane is thoroughly dried.

(Nota: Confirm that absolute ethanol has been added to Wash Buffer 1. The presence of ethanol has a serious impact on subsequent experiments, so membrane drying is crucial. Dopo la centrifugazione, ensure there is no ethanol present before elution. Discard the waste liquid and the collection tube.

After washing with Wash Buffer 1, the membrane on the RNA extraction purification column should only have a slight color. Dopo la centrifugazione, carefully remove the RNA extraction purification column without touching the collection tube to ensure no ethanol interference.)

9. Place the RNA extraction purification column into a new centrifuge tube, drip 100 μl Elution Buffer onto the membrane, incubate at room temperature for 5 minuti (15°C~25°C), centrifuge at more than 8,000 giri al minuto per 1 minuto.

(Nota: Eluting RNA with 50 μl Elution Buffer can increase RNA concentration but decrease total RNA yield.)

10. Repeat the previous step.

(Nota: A new centrifuge tube can be used to collect the RNA eluted the second time, or the original collection tube can be used to continue collecting RNA.)