Polysaccharide and Polyphenol Plant Total RNA (Plus) Small Quantity Extraction Kit

1. Komponente kompleta reagensa

Tehnički podaci	50T	100T
Mačka. Ne.	SN0305AD	SN0306AD
RNA Extraction Columns (set)	50 (set)	100 (set)
DNA Clean-Up Columns (set)	50 (set)	100 (set)
RNA Extraction Buffer I	30 ml	2×30 ml
Inhibitor Removal Buffer	30 ml	2×30 ml
Pufer za pranje 1	15 ml	2×15 ml
Elucijski pufer	20 ml	20 ml
Priručnik za upute	1	1

 

2. Skladištenje

This reagent kit can be stored at room temperature (15-25℃) in a dry environment and is stable 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

This RNA purification kit provides a fast and efficient method for purifying polysaccharide and polyphenol plant total RNA, suitable for most polysaccharide and polyphenol-rich plant tissues. In general, plant tissues rich in polysaccharides and polyphenols contain a high level of secondary phenolic metabolites and polysaccharides, significantly affecting RNA extraction efficiency. This kit can effectively remove polysaccharides and polyphenols, as well as efficiently eliminate DNA contamination from samples. If the experiment is sensitive to DNA, it is recommended to use primers spanning introns for downstream experiments.

The RNA Fast Purification Kit can extract total RNA from plant tissues (including nuclear RNA and cytoplasmic RNA) within 1 sat. The extracted RNA can be directly used for RT-PCR, Northern blotting, itd. Cijeli postupak pročišćavanja ne zahtijeva toksične reagense kao što je fenol-kloroform, making this RNA purification kit suitable for various other sample types.

5. Eksperimentalni principi i postupci

6. Ekstrakcijski postupak

Precautions before starting the experiment:

A. Before using Pufer za pranje 1, add the specified amount of absolute ethanol according to the label on the reagent bottle, and check the box on the label to indicate that absolute ethanol has been added.

B. The Elution Buffer is a 0.1X TE otopina containing minimal EDTA. If EDTA affects subsequent experiments, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Sample Processing:

A. Material Collection and Storage:

If fresh materials cannot be used immediately, place them in liquid nitrogen and store them at -80°C.

B. Whenever possible, collect fresh materials, as they contain fewer polysaccharides and polyphenols.

2. Grind approximately 100 mg of fresh samples or not more than 20 mg of dry material using liquid nitrogen.

3. Dodati 600μl RNA Extraction Buffer I, ensuring there are no blocky tissues in the ground sample. Blocky tissues are difficult to lyse and can reduce RNA yield.

4. Vortex for 30 sekundi.

5. Centrifuge the lysate for 5 minutes at 12,000 rpm.

(Bilješka: Polysaccharide plant materials may produce a lot of sticky substances at this step, which can shear RNA in subsequent steps. Stoga, remove these substances during this step.)

6. Transfer the supernatant obtained in the previous step to a DNA Clear Purification Column, centrifuga na 12,000 RPM za 30 sekundi, and collect the filtrate (Bilješka: RNA is present in the filtrate).
7. Dodati 250μl of absolute ethanol, mix by pipetting. If there is a small amount of precipitation, it does not affect subsequent experiments. Add the liquid to the RNA purification column, centrifuga na 12,000 RPM za 30 sekundi, and discard the flow-through.
8. Dodati 700μl Inhibitor Removal Buffer to the RNA purification column, centrifuga na 12,000 RPM za 30 sekundi, and discard the flow-through.
9. Dodati 700μl Wash Buffer 1 to the RNA purification column, centrifuga na 12,000 RPM za 30 sekundi, and discard the flow-through.
10. Dodati 500μl Wash Buffer 1 to the RNA purification column, centrifuga na 12,000 RPM za 3 minuta, and discard the flow-through.
11. Place the RNA purification column into a new 1.5ml centrifuge tube, air-dry the membrane at room temperature for 2 minuta.

(Bilješka: Confirm the addition of ethanol to Wash Buffer 1. The presence of ethanol significantly affects subsequent experiments. Stoga, membrane drying is crucial. After centrifugation, ensure the absence of ethanol before elution. Discard the waste and the collection tube. After using Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. Carefully remove the RNA purification column after centrifugation, ensuring it does not touch the collection tube to avoid ethanol contamination.)

12. Drip 50-100μl Elution Buffer onto the membrane, centrifuga na 12,000 RPM za 1 minuta, and collect the RNA solution.

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