Molecular biology related experimental steps and precautions (1)

Molecular biology related experimental steps and precautions (1)

The friends who just entered the lab, when you are doing RNA extraction, RT-PCR and QPCR experiments, will there be such a feeling, obviously done according to the experimental steps, the experimental results are not satisfactory, even very Bad? Xiaobian I have also been abused by experiments~ In order to reduce the impact of the experiment on the pro, I will present some small experiences in the experiment process, hoping to help your friends~

(1) RNA extraction articles

In the process of RNA extraction, strict prevention of RNase contamination and try to inhibit its activity are the key to the success of this experiment. For the extraction of human, animal and plant tissues, bacteria, cells and other RNAs, the Trizol method works well to separate RNA/DNA/protein in a sample at the same time. Trizol lyses cells, lyses cell contents, and maintains RNA integrity by containing RNase inhibitors.

RNA extraction kits are also available if the purity of the RNA is extremely high or the sample is difficult to extract. Some students have used the domestic roots, and the effect is good. Xiaobian has used Omega's RNA extraction kit, and the effect is also very good. Promega's total RNA extraction kit is also good for small amount of purification of various samples.

Total RNA extraction from plants and animals - The specific steps of the Trizol method are as follows:

1. After the tissue is ground, 50-100 mg is added to 1 ml of Trizol, and mixed and lysed. The total volume of the sample should preferably not exceed 10% of the volume of Trizol used.
2. After the slurry was allowed to stand at room temperature for 5-10 min, add 200 μl of chloroform, cover the EP tube, and shake it on a vortex shaker for 15 s. Then let stand on ice for 10 min.
3. Centrifuge at 10000 g for 15 min at 4 ° C, pipette 500 μl of the supernatant clear liquid into another clean EP tube, add an equal volume (500 μl) of isopropanol, mix gently and let stand for 10 min.
4. Centrifuge at 10,000 g for 10 min at 4 ° C to remove the supernatant. After centrifugation, a gelatinous white precipitate may appear on the tube wall or at the bottom of the tube. Care should be taken to avoid the loss of sediment.
5. Add 1 ml of pre-cooled 75% absolute ethanol (DEPC water configuration) to the EP tube and gently bounce the pellet with your fingers, then centrifuge at 10,000 g for 5 min at 4 °C.
6. Carefully discard the supernatant and let it stand at room temperature for 2-5 minutes. Be careful not to overdo it.
7. Dissolve the RNA in 20-50 μl of DEPC water, if necessary, in a water bath at 55-60 ° C for 3-5 min. The EP tube cover needs to be sealed during the water bath. The extracted RNA can be used in subsequent experiments or stored at -80 °C.

Precautions:

1. Before the extraction, all the required experimental items were placed in a clean bench, and the table top was sprayed with RNase water for 15 minutes.
2. Wear masks and gloves during the extraction process, and frequently change gloves (disposable gloves).
3. During the extraction process, people should avoid walking around the clean bench to avoid RNase smashing into the ultra-clean platform.
4, the sample should be fully homogenized and lysed, otherwise it will cause low RNA extraction.
5. In step 3, the centrifuge tube should be light when it is taken out of the centrifuge, so as to avoid the material in the tube from oscillating and causing the lower layer to precipitate. When sucking the supernatant, it must be light, avoid taking too much, a small amount can be, sucking 400-500μl is OK, and then it is easy to hit the lower layer of precipitation.
6. In step 5, the sediment must be bounced off, allowed to float in alcohol, and then placed for 1-2 minutes, allowing the alcohol to fully contact the precipitate, fully dissolve the organic reagent, and then centrifuge. But sometimes it also causes some impurities to settle.
7. The last step must be sufficient to dissolve the RNA. The amount of tissue extracted is still quite large, generally about 50μl.

There are two other experiences in molecular biology to share with you, so stay tuned!

Supplier Extract Powder

We're Professional Supplier Extract Powder manufacturers and suppliers in China specialized in providing high-quality products at low price. We warmly welcome you to buy or wholesale bulk Supplier Extract Powder for sale here from our factory. For a free sample, contact us now.