TRIzol Reagent

Self-provided reagents

Chloroform, isopropanol, 70% ethanol (DEPC water configuration), Rnase Free H₂O.

Ⅰ Preparation before experiment

The key to RNA preparation is to inhibit RNA degrading enzymes in cells and prevent contamination of RNA degrading enzymes in the equipment and reagents used. Therefore, the following measures must be taken in the experiment: wear disposable clean gloves; use a special experimental bench for RNA operation; avoid talking during the operation, etc. The above methods can prevent the contamination of the experimenter's sweat and saliva by RNA degrading enzymes.

Cautions:
1. Try to use disposable plastic utensils. If glassware is used, it should be treated with 0.1% DEPC aqueous solution at 37°C for 12 hours before use, and then autoclaved at 120°C for 30 minutes to remove residual DEPC.
2. Reagents used for RNA experiments must be sterilized by dry heat (180°C, 60min) or in glass containers after DEPC water treatment and sterilization using the above methods (disposable plastic containers for RNA experiments can also be used) , The sterile water used must be treated with 0.1% DEPC and then autoclaved.
3. Reagents and sterile water for RNA experiments should be dedicated to avoid cross-contamination after mixing.

Ⅱ Experimental operation

The usage of TRIzol Reagent is as follows

Sample size and RNA yield

TRIzol usage instructions

Common problems

1. Low extraction rate. Possible reasons: (a. Sample lysis or homogenization is not complete; b. RNA precipitation is not completely dissolved)
2.A260/A280<1.65. Possible reasons: (a. When measuring the absorbance, the RNA sample was not dissolved in water, but dissolved in TE; b. The amount of tissue added when the sample was homogenized was too much; c. After stratification, the supernatant was less than 500μl; d. The organic phase was mixed in the water phase)
3. Excessive DNA contamination. Possible reasons: (a. The amount of reagents added during sample homogenization is too small or the amount of tissue is too much; b. The sample contains organic solvents) Solution: using this reagent usually genomic DNA contamination content <0.1ng/μl, if it is necessary to remove DNA contamination completely, please use Rnase Free DNase I (HaiGene: A3001) to digest and remove genomic DNA contamination. If you use the Gold Medal cDNA First Strand Reverse Transcription Kit (HaiGene: D0401) (HaiGene: D0401), there is no need to digest to remove genomic DNA contamination in advance. The kit contains reagents to remove genomic DNA contamination.