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

1. Bestandteile des Reagenzienkits

2. Lagerung

This reagent kit can be stored at room temperature (15-25℃) in a dry environment and is stable for 12 Monate.

3. Anweisungen zur Verwendung des Reagenzienkits

3.1 Dieses Kit ist für molekularbiologische Forschungszwecke bestimmt und sollte nicht zur Diagnose oder Behandlung von Krankheiten verwendet werden.

3.2 Einige Bestandteile des Kits enthalten Reizstoffe; Es ist ratsam, die erforderlichen Vorsichtsmaßnahmen zu treffen (wie das Tragen von Schutzkleidung und Schutzbrillen).

3.3 Die Verwendung dieses Kits erfordert zusätzliche Ausrüstung wie eine Hochgeschwindigkeitszentrifuge, Wasserbad (Metallbad), Vortexmixer, Wasserfreies Ethanol, flüssiger Stickstoff, Chloroform, steriles entionisiertes Wasser, und EP-Röhren.

4. Einführung in das Reagenzienkit

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 Stunde. The extracted RNA can be directly used for RT-PCR, Northern blotting, usw. The entire purification process does not require toxic reagents such as phenol-chloroform, making this RNA purification kit suitable for various other sample types.

5. Experimentelle Prinzipien und Verfahren

6. Extraktionsprozess

Vorsichtsmaßnahmen vor Beginn des Experiments:

A. Before using Waschpuffer 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-Lösung mit minimalem EDTA-Gehalt. Wenn EDTA nachfolgende Experimente beeinflusst, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Probenverarbeitung:

A. Materialsammlung und -lagerung:

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 Trockenmaterial mit flüssigem Stickstoff.

3. Hinzufügen 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 Sekunden.

5. Zentrifugieren Sie das Lysat 5 Minuten um 12,000 U/min.

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

6. Transfer the supernatant obtained in the previous step to a DNA Clear Purification Column, Zentrifuge bei 12,000 U/min für 30 Sekunden, and collect the filtrate (Notiz: RNA is present in the filtrate).
7. Hinzufügen 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, Zentrifuge bei 12,000 U/min für 30 Sekunden, and discard the flow-through.
8. Hinzufügen 700μl Inhibitor Removal Buffer to the RNA purification column, Zentrifuge bei 12,000 U/min für 30 Sekunden, and discard the flow-through.
9. Hinzufügen 700μl Wash Buffer 1 to the RNA purification column, Zentrifuge bei 12,000 U/min für 30 Sekunden, and discard the flow-through.
10. Hinzufügen 500μl Wash Buffer 1 to the RNA purification column, Zentrifuge bei 12,000 U/min für 3 Protokoll, 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 Protokoll.

(Notiz: Confirm the addition of ethanol to Wash Buffer 1. The presence of ethanol significantly affects subsequent experiments. daher, membrane drying is crucial. Nach Zentrifugation, ensure the absence of ethanol before elution. Entsorgen Sie den Abfall und das Sammelröhrchen. 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. Tropfen 50-100μl Elution Buffer auf die Membran, Zentrifuge bei 12,000 U/min für 1 Minute, and collect the RNA solution.

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