Набор для мелкомасштабного выделения геномной ДНК растений методом CTAB

1. Компоненты набора реагентов

2. Хранилище

This kit should be stored at room temperature (15-25℃) in dry conditions and can be stored for 12 месяцы. DNA extraction purification columns can be stored in a cool and dry environment for up to 1 год. RNase A contains a preservative and can be transported at room temperature, но для длительного хранения, it should be kept at -20℃.

3. Инструкции по использованию набора реагентов

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, водяная баня (металлическая ванна), вихревой смеситель, безводный этанол, liquid nitrogen, chloroform, стерильная деионизированная вода, and EP tubes.

4. Знакомство с набором реагентов

The СТАБ-based Plant Очистка ДНК Kit provides an improved CTAB method for purifying DNA, utilizing a specific binding buffer that efficiently precipitates DNA, and subsequently collects high-purity DNA through an adsorption column.

This kit is widely used for plant tissues and fungi, capable of extracting total DNA from samples within 2 часы (including mitochondrial DNA and chloroplast DNA). The extracted DNA can be directly used for downstream experiments such as ПЦР, Саузерн-блоттинг, и другие.

5. Экспериментальные принципы и процедуры

6. Процесс экстракции

Precautions before starting the experiment:

1. Reagent buffers A and C может выпадать в осадок в условиях низких температур. It is recommended to heat at 65°C for 5 minutes and use after the precipitates have dissolved.
2. СтиратьБуфер 1 should have the specified amount of anhydrous ethanol added as indicated on the bottle label. Mark the label once ethanol has been added.
3. Elution buffer is a 0.1х ТЕ решениеcontaining minimal EDTA. If EDTA affects subsequent experiments, sterile deionized water is recommended as a substitute for the elution buffer.

1. Обработка образцов:
2. Material Collection and Storage:

Freshly collected material, if not immediately used, should be placed in liquid nitrogen and ultimately stored at -80°C. Dried materials can be stored at room temperature.

1. Если возможно, collect fresh material as it contains fewer polysaccharides and polyphenols.
2. When collecting fungi from liquid culture, separate the liquid by centrifugation and collect the fungal bodies.
3. Grind around 100 мг свежих проб или не более 20 mg of dry material using liquid nitrogen.

(Примечание: Different sample quantities may require optimization through preliminary experiments before use.)

3. Добавлять 550 μl of reagent buffer A and 10 μl of RNase A (10 мг/мл) to ensure there are no tissue clumps in the ground sample. Tissue clumps are difficult to lyse and can reduce DNA yield. Do not mix reagent buffer A and RNase Aперед использованием.
4. Инкубируйте при 65°C в течение 20-30 минуты, gently invert 2-3 раз. This step is for cell lysis.
5. Centrifuge the lysate for 5 minutes at 14,000 об/мин (20,000×г).

(Примечание: Some plant materials may have a lot of sticky substances at this step, which can shear DNA in subsequent steps. Ideally, remove these substances by transferring the supernatant to a new centrifuge tube after centrifugation.)

6. Carefully transfer the liquid obtained in the previous step to a new centrifuge tube.

(Примечание: Approximately 500 μl of liquid can be transferred; for some species, it may be less than 500 мкл.)

7. Add an equal volume of chloroform to the lysate and gently invert to mix.

(Примечание: Например, добавлять 500 μl of chloroform if you have 500 μl of lysate. If the lysate volume is less than 500 мкл, adjust the chloroform volume accordingly.)

8. Центрифуга в 12,000 об/мин для 10 минуты.
9. Carefully transfer the supernatant to a new centrifuge tube (approximately 500 мкл).
10. Добавьте равный объем reagent buffer C and an equal volume of anhydrous ethanol to the lysate, and mix.

(Например, if you add 450 μl of reagent buffer C, затем добавьте 450 μl of anhydrous ethanol. If the lysate volume is less than 450 мкл, reduce the amount of reagent buffer C proportionally. Some precipitation will occur upon adding reagent buffer C, but it will not affect subsequent experiments.)

11. Transfer the obtained liquid to a DNA purification column (набор), approximately 650-700 μl each time. Centrifuge at over 8,000 об/мин для 1 минута, discard the collected waste, and reinsert the collection tube into the purification column for the next step.
12. Повторить шаг 11, adding the remaining liquid to the DNA purification column (набор) and centrifuge at over 8,000 об/мин для 1 минута. Discard the waste and the collection tube.
13. Place the DNA purification column (набор) into a new collection tube, добавлять 300 мкл Стирать Буфер 1, центрифуга при более чем 8,000 об/мин для 1 минута, выбросить отходы, and reinsert the DNA purification column (набор) into the tube for the next step.

(Примечание: Ensure that anhydrous ethanol has been added to Стирать Буфер 1.)

14. Добавлять 500 μl of Rinse Buffer 1 to the DNA purification column (набор), центрифуга в 14,000 об/мин (20,000×г) для 2 минуты, slightly prolong the centrifugation time for a drier membrane.
15. Place the DNA purification column (набор) в новую центрифужную пробирку, открыть, и нагреваем при температуре 65°С в течение 2 минуты. This step may be prolonged to evaporate ethanol as much as possible to prevent residual ethanol from affecting downstream experiments.
16. Капать 100 μl of elution bufferна мембрану, центрифуга в 12,000 об/мин для 2 минуты.

(Примечание: 1. Элюирование ДНК с помощью 50 μl elution buffer can increase DNA concentration but decrease total DNA yield. 2. The eluate can be reapplied to the DNA purification column for a second elution, центрифуга в 12,000 об/мин для 2 minutes to collect, which may improve DNA yield.)