Stool Total RNA Extraction Kit

1. Komponente kompleta reagensa

Tehnički podaci	50T	100T
Mačka. Ne.	SN0341	SN0342
RNA Extraction Columns (set)	50 (set)	100 (set)
Humic Acid Removal Reagent I	50ml	2x50ml
Reagent Buffer C Plus	30 ml	2x30ml
Inhibitor Removal Buffer	30ml	2x30ml
Lizocim	1ml	2x1ml
Proteinaza K	1ml	2x1ml
Pufer za pranje 1	15 ml	2 × 15 ml
Elucijski pufer	20 ml	20 ml
Priručnik za upute	1	1

2. Skladištenje

Ovaj se komplet reagensa treba čuvati na sobnoj temperaturi (15-25°C) under dry conditions and can be kept for 12 mjeseca. Lysozyme and Proteinase K contain preservatives, allowing for transportation at room temperature. Za dugotrajno skladištenje, it should be stored at -20°C.

3. Upute za korištenje kompleta reagensa

3.1 This kit is intended for molecular biology research purposes and should not be used for disease diagnosis or treatment.

3.2 Some components in the kit contain irritants; it is advisable to take necessary precautions (such as wearing protective clothing and goggles).

3.3 The use of this kit requires additional equipment such as a high-speed centrifuge, vodena kupelji (metalna kupka), vrtlog miksera, bezvodni etanol, liquid nitrogen, chloroform, sterilna deionizirana voda, and EP tubes.

4. Uvod u komplet reagensa

The Fecal Total RNA Purification Kit provides a fast and efficient method for purifying total RNA from feces and vomit, widely applicable to the extraction of Gram-positive bacteria, Gram-negative bacteria, and viral RNA in feces and vomit samples.

This kit effectively removes impurities and inhibitors from feces and vomit, reducing inhibition in subsequent downstream experiments such as PCR. The extracted RNA can be directly used for PCR, Južni blot, itd. Cijeli postupak pročišćavanja ne zahtijeva toksične reagense kao što je fenol-kloroform, making the RNA purification kit suitable for various other sample types.

5. Eksperimentalni principi i postupci

6. Ekstrakcijski postupak

Precautions before starting the experiment:

A. Prior to use, Dodajte navedenu količinu bezvodnog etanola u PranjeBuffer 1 as indicated on the reagent bottle label, and mark with a check on the label to indicate the addition of anhydrous ethanol.

B. Buffer za eluciju je a 0.1X TE otopina containing a minimal amount of EDTA. If EDTA may affect subsequent experiments, it is recommended to substitute Elution Buffer with sterile deionized water.

1. Sample Processing (Inhibitor Removal):

A: Add approximately 200mg of samples such as feces, add 1ml of Humic Acid Removal Reagent I, vortex thoroughly for 1-2 minuta, centrifuga na 12,000 RPM za 2 minuta, and discard the supernatant.

B: If the extracted sample is a virus sample, you can proceed directly to step 2. If the sample contains a high amount of inhibitors, it is recommended to perform the inhibitor removal step first, although it may reduce the yield of viral nucleic acid.

2. Sample Lysis:

A: For extracting nucleic acids from bacterial samples, dodati 560μl of Buffer C Plus, 20μl Proteinase K, and 20μl Lysozyme to the centrifuge tube from the previous step. Invert and mix thoroughly, digest at 85℃ for 5 minuta, inverting the tube 6-7 times during digestion.

B: For extracting nucleic acids from virus samples, dodati 560μl of Buffer C Plus and 20μl Proteinase K. Digest at 85℃ for 5 minuta, inverting the tube 6-7 times during digestion.

3. Centrifuga na 12,000 RPM za 5 minuta, transfer the supernatant to a new centrifuge tube (approximately 500μl liquid transferred).
4. Add an equal volume of anhydrous ethanol, Temeljito pomiješajte. Precipitation may occur, but it does not affect subsequent experiments.
5. Transfer the obtained liquid to an RNA purification column (approximately 650-700μl each time), incubate at room temperature for 2 minuta, centrifuge at over 8,000 RPM za 1 minuta, discard the collected waste, and reinsert the collection tube into the purification column for the next step.
6. Place the RNA purification column into a new collection tube, dodati 400μl of Inhibitor Removal Buffer, centrifuge at over 8,000 RPM za 1 minuta, odbaciti otpad, and place the nucleic acid purification column back into the tube for the next step.
7. Dodati 500μl pufera za pranje 1to the RNA purification column, centrifuga na 14,000 rpm (20,00×g) za 2 minuta, extending the centrifugation time as needed to ensure membrane drying.

(Bilješka: The presence of ethanol has a serious impact on subsequent experiments, so membrane drying is crucial. After centrifugation, ensure no ethanol is present before elution, then discard the waste and collection tube.)

8. Place the RNA purification column into a new centrifuge tube, dodati 30μl – 50 μl pufera za elucijuto the membrane, incubate at room temperature for 5 minuta (15℃-25℃), centrifuge at over 8,000 RPM za 1 minuta.

(Bilješka: Using 30μl of Elution Buffer to elute RNA can increase RNA concentration but may reduce total RNA yield.)

9. Repeat the previous step.

(Bilješka: A new centrifuge tube can be used to collect the RNA eluted in the second wash or continue using the original collection tube to collect RNA.)