Total RNA extraction reagent (Trizol method) instruction manual

"Ordering Information"
Catalog Number Product Name Specifications
MTO-1009 2×PCR TaqMix (with dye) 1ml
MTO-1010 2×PCR TaqMix (no dye) 1ml
MTQ-1001 2×qPCR TaqMix 1ml
41003 GelRed Nucleic Acid Dyes 500ul
31000 20×EvaGreen 1ml
DDLK-010 DIG DNA PCR Labeling Kit 10T
RDLK-010 Random Primer DIG Labeling Kit 10T
DIGD-110 DIG Hybridization Test Kit I (NBT/BCIP method) 10T
DIGD-210 DIG Hybridization Test Kit II (CDP-Star Method) 10T
Hyb-50, Hyb-100 Hyb High-efficiency Hybrid Solution 50ml, 100ml

Product No:MRN0210

MyLab® Total RNA Extraction Reagent (for animal tissues/plant tissues, insects and cells)
(Trizol method)
user's Guide

Specification Save Validity
100ml 2-8°C 12 months "Uses and Features"
1. This kit can be used for rapid extraction of almost all fresh or liquid nitrogen-preserved animal and plant tissues, insects and cellular RNA with good versatility.
2. No DNase, RNase inhibitor, etc. are required.
3. The required sample volume is small and can be adjusted according to the amount of starting material.
4. Easy to operate, the whole process <1h.
5. The extracted RNA is high in purity, free from protein and DNA contamination, and can be used in downstream experiments such as RT-PCR amplification, Northern blot analysis, dot blot hybridization, Poly(A)+ selection, in vitro translation, RNase without additional treatment. Protection experiments and molecular cloning, etc.
"Self-prepared reagents"
Isopropanol; 75% ethanol without RNase; H2O with DEPC; 3M sodium acetate; chloroform.
"Precautions"
1. Protect the operating environment, RNase contamination of container consumables and reagents used. The instruments and consumables used were RNase-free. Change gloves during the operation.
2. Too much or too little may affect the quality or yield of RNA. If the amount of starting material is small, the RNA is expected to have a very low yield. When isopropanol is precipitated, 0.5~1 μl of 20 mg/ml hepatic glycogen solution can be added to assist RNA precipitation.
3. Dissolve RNA in TE buffer (between pH 7.5 and 8.2) to detect its light absorption at OD260. Do not dilute RNA in distilled water or DEPC water to detect OD260.
4. Do not touch the skin or swallow directly to avoid burns. If contact with skin, rinse immediately with a mild detergent and plenty of water. Avoid rubbing with ethanol, ethanol will increase the degree of burns. If you feel unwell, please seek medical help immediately.
5. This product is for scientific research use only. Do not use in medicine, clinical treatment, food, etc.
"Steps"
1. Lysis treatment:
1 Animals/plant tissues, insects:


25-100 mg of liquid nitrogen frozen or fresh tissue, placed in a mortar containing liquid nitrogen, and immediately ground to a powder (it is best to always have liquid nitrogen in the mortar during the grinding process). After the research, directly add 1 ml of total RNA extraction reagent to the mortar and continue grinding to a powder. Weigh 2 cells
1) Suspension cultured cells:
The cells were collected by centrifugation and 1 ml total RNA extraction reagent was added per 5-10 x 106 animals, plants and yeast cells. It is best not to wash the cells before adding the total RNA extraction reagent to avoid increasing the possibility of mRNA degradation and RNase contamination.
2) adherent cultured cells:
The cells were directly lysed by adding total RNA extraction reagent to the culture plate, and 1 ml of total RNA extraction reagent was added per 10 cm 2 area. Blow several times with the sampler.
Note: The amount of total RNA extraction reagent is determined by the area of ​​the culture dish, not by the number of cells. If the total RNA extraction reagent is insufficient, it may cause DNA contamination in the extracted RNA. If the lysate is very viscous when sucked up with a pipette tip, it is often necessary to add a little more total RNA extraction reagent.
2. Allow 5-10 minutes at room temperature to fully lyse, and then melt into a liquid and then transfer to a 1.5 ml centrifuge tube without RNase (after grinding into a powder, until the room temperature is fused into a liquid state, it can no longer be stirred with a grinding crucible, otherwise it will Produce DNA contamination). Note: At this time, it can be placed at -70 °C for a long time.
3. Transfer the above liquid into a 1.5 ml centrifuge tube, add 200 μl of chloroform to it, and cover the tube. Mix vigorously and shake for 5 min at room temperature. Note: The vortex oscillator is disabled to avoid genomic DNA breaks.
4. Centrifuge at 12,000 rpm for 15 min at 4 °C. The sample is divided into three layers: the bottom layer is a pink organic phase, and the upper layer is a colorless aqueous phase and an intermediate layer. RNA is mainly present in the aqueous phase.
5. Carefully pipette the upper aqueous phase into another centrifuge tube. Note: Never draw the intermediate interface.
6. Add an equal volume of isopropanol and 1/10 volume of 3M sodium acetate to the centrifuge tube, gently mix upside down and place at -20 °C for 15-20 min.
7. Centrifuge at 14000 rpm for 15 min at 4 ° C. Pour off the supernatant and take care not to touch the pellet. RNA precipitation is often invisible prior to centrifugation and forms a gelatinous precipitate on the tube side and bottom of the tube after centrifugation.
8. Add 200 ul of 75% ethanol, gently shake the tube and suspend the pellet. The supernatant was discarded by centrifugation at 4 ° C for 2-3 min. Try to discard the supernatant.
9. Place the tube on the clean bench and let it dry for about 2-3 minutes (do not dry completely). Add 30-50 μl of RNase-free water. Shake for 30 sec and centrifuge instantaneously.
10. Transfer the extracted RNA immediately to a downstream experiment or store at -70 °C.