Krv Ukupni komplet za ekstrakciju RNA (Uklanjanje genomskog DNK)

1. Komponente kompleta reagensa

2. Skladištenje

Ovaj se komplet reagensa treba čuvati na sobnoj temperaturi (15-25℃) in a dry environment and can be preserved for 12 mjeseca.

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 RNA purification kit provides a fast and efficient method for purifying RNA from blood and other fluid tissues. It is suitable for most biological fluids and tissues. This RNA purification kit can be applied to blood samples exceeding 0.5 ml, and through DNA removal technology, the extracted RNA is virtually free of genomic DNA.

The RNA Fast Purification Kit allows for the extraction of total RNA (including nuclear RNA and cytoplasmic RNA) od krvi iznutra 2 sati. The purified RNA can be directly used for applications such as RT-PCR, Northern blotting, itd. Cijeli postupak pročišćavanja ne zahtijeva toksične reagense kao što je fenol-kloroform, making the RNA purification kit well-suited for various other sample types.

5. Eksperimentalni principi i postupci

6. Ekstrakcijski postupak

Precautions before starting the experiment:

A. Prije upotrebe, add the specified amount of ethanol to Wash Buffer 1 as indicated on the reagent bottle label, and mark a check on the label to indicate the addition of ethanol.

B.Elution Buffer is a 0.1x TE solution containing a minimal amount of EDTA. If EDTA affects subsequent experiments, it is recommended to use sterile deionized water as a substitute for the Elution Buffer.

1. Sample Processing: Take 200μL of blood into an EP tube, dodati 80μL of Red Blood Cell Lysis Buffer 10x, and mix thoroughly by pipetting.
2. Dodati 500μL RNA Extraction Buffer 1 plus, vigorously shake to mix, centrifuge at 12000rpm for 3 minuta.
3. Transfer the supernatant to a DNA purification column, centrifuge at 12000rpm for 1 minuta.
4. Dodati 250μL of ethanol to the supernatant, pipette and mix, transfer the liquid to the RNA purification column, centrifuge at 12000rpm for 30 sekundi, discard the supernatant.
5. Dodati 600μL Inhibitor Removal Buffer to the RNA purification column, centrifuge at 12000rpm for 30 sekundi, discard the supernatant.
6. Dodati 600μL Wash Buffer 1 to the RNA purification column, centrifuge at 12000rpm for 30 sekundi, discard the supernatant.

(Bilješka: Confirm that ethanol has been added to Wash Buffer 1. Ethanol presence significantly affects subsequent experiments. Stoga, membrane drying is crucial. After centrifugation, ensure no ethanol remains before elution, then discard the waste and collection tube. After using Wash Buffer 1, the membrane on the RNA purification column should have a slight color. After centrifugation, carefully remove the RNA purification column, ensuring it does not touch the collection tube to prevent ethanol interference.)

7. Repeat step 6.
8. Centrifuge the empty tube at 12000rpm for 2 minuta (to evaporate residual ethanol).
9. Place the RNA purification column in a new centrifuge tube, drip 100μL Elution Buffer onto the membrane, incubate at room temperature for 5 minuta (15℃~25℃), centrifuge at 12000rpm for 1 minuta.

(Bilješka: Eluting RNA with 50μL Elution Buffer can increase RNA concentration but reduce total RNA yield.)