Бактериальная тотальная РНК (плюс) Комплект для извлечения

1. Компоненты набора реагентов

2. Хранилище

Этот набор реагентов следует хранить при комнатной температуре. (15-25℃) in a dry environment and can be preserved for 12 месяцы. Lysozyme contains a preservative, allowing for transportation at room temperature; однако, for long-term storage, it should be stored 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

This RNA purification kit provides a rapid and efficient purification solution for bacterial RNA, suitable for most bacterial tissues. The kit employs DNA removal technology to effectively eliminate genomic DNA, and the resulting RNA samples typically do not require on-column digestion, reducing the risk of RNA degradation.

The RNA Fast Purification Kit allows for the extraction of total bacterial RNA (including nuclear RNA and cytoplasmic RNA) в пределах 1 час. The extracted RNA can be directly used for RT-ПЦР, Нозерн-блоттинг, и т. д.. The entire purification process does not involve toxic reagents such as phenol-chloroform, making the RNA purification kit well-suited for various sample types.

5. Экспериментальные принципы и процедуры

6. Процесс экстракции

Precautions before starting the experiment:

А. Перед использованием, add the specified amount of absolute ethanol to Промывной буфер 1 as indicated on the reagent bottle label, and mark with a check to indicate the addition of absolute ethanol.

Б. Элюирующий буфер представляет собой 0.1х ТЕ решение содержащий минимальное количество ЭДТА. If EDTA may impact subsequent experiments, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Образец обработки:

А. Gram-positive bacteria: Centrifuge the bacterial suspension at 4℃, 12,000 об/мин для 2 минуты, collect bacterial cells (1.53 мл), выбросить супернатант, добавлять 100мкл Лизоцим (10мг/мл), thoroughly mix the bacterial cells, and incubate at room temperature for 15-30 минуты.

Б. Gram-negative bacteria: Centrifuge the bacterial suspension at 4℃, 12,000 об/мин для 2 минуты, collect bacterial cells (1.53 мл), выбросить супернатант, добавлять 10мкл Лизоцим (10мг/мл), thoroughly mix the bacterial cells, and incubate at room temperature for 3-5 минуты.

2.Добавлять 400μl RNA Extraction Buffer I, vortex to mix thoroughly.

3. Transfer the lysate to a DNA clearance purification column, центрифуга в 13,000 об/мин для 2 минуты, collect the filtrate (RNA is present in the filtrate).

4. Accurately estimate the volume of the filtrate, добавлять 0.5 times the volume of absolute ethanol, тщательно перемешать; if precipitation occurs, it does not affect subsequent experiments.

5. Add the obtained liquid to the RNA extraction purification column (approximately 650~700μl each time), centrifuge at more than 8,000 об/мин для 1 минута, выбросить собранную отработанную жидкость, and reinsert the collection tube into the RNA extraction purification column for the next step.

6. Повторить шаг 5, add the remaining liquid to the RNA extraction purification column, centrifuge at more than 8,000 об/мин для 1 минута, выбросьте отработанную жидкость и трубку для сбора.

7. Place the RNA extraction purification column into a new collection tube, добавлять 600мкл буфера для удаления ингибитора, centrifuge at more than 8,000 об/мин для 1 минута, выбросить отработанную жидкость, and reinsert the RNA extraction purification column into the tube for the next step.

8. Добавлять 700мкл промывочного буфера 1 to the RNA extraction purification column, центрифуга в 14,000 об/мин (20,000×г) для 2 минуты, extend the centrifugation time appropriately to ensure the membrane is thoroughly dried.

(Примечание: Confirm that absolute ethanol has been added to Wash Buffer 1. The presence of ethanol has a serious impact on subsequent experiments, so membrane drying is crucial. После центрифугирования, ensure there is no ethanol present before elution. Discard the waste liquid and the collection tube.

After washing with Wash Buffer 1, the membrane on the RNA extraction purification column should only have a slight color. После центрифугирования, carefully remove the RNA extraction purification column without touching the collection tube to ensure no ethanol interference.)

9. Place the RNA extraction purification column into a new centrifuge tube, drip 100 μl Elution Buffer onto the membrane, incubate at room temperature for 5 минуты (15°C~25°C), centrifuge at more than 8,000 об/мин для 1 минута.

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

10. Repeat the previous step.

(Примечание: A new centrifuge tube can be used to collect the RNA eluted the second time, or the original collection tube can be used to continue collecting RNA.)