(1) A sample of 3 g was taken, and 10% of the sample weight of poly-polypyrrololone (PVPP) was added, and the mixture was ground to a powdery state with liquid nitrogen, and the powder was divided into about 3 g per part and stored at -80 °C.
(2) Participate in 20ml in a 50ml falcon tube to obtain a mitigation fluid. The group in which the liquid was taken out was changed to 0.2 mol/L sodium dehydrated sodium tetraborate (sodiumtetriboratedecahydrate), 30 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% (W/V) sodium dodecyl sulfate (SDS), 1%. Deoxycholate sodium salt. Further, 0.0309 g of dithiothreitol (DTT), 200 ul of ethyl phenyl polyglycol (NP-40) and 0.4 g of polyethylene pyrrolane ketone (PVP-40) were added and equilibrated at 80 ° C for 15 min. The samples were taken at -80 ° C, and after the thermal equilibration, the mitigation liquid was obtained and the vibration was mixed. Then participate in 800 ul of proteinase K (20 mg / ml), cover the tube cover, lay flat on a shaker with a speed of 120 r / min and a temperature of 42 ° C, and swing to obtain 1.5 h.
(3) After this, 1.6 ml of 2 mol/L KCl was added to the sample tube, and the mixture was shaken, and the sample tube was placed at 4 ° C for 1 to 1.5 hours. The extract solution was transferred to a glass centrifuge tube and centrifuged at 12000 x g for 20 min at 4 °C. The supernatant was placed in a new glass centrifuge tube and subjected to 1/4 (or 1/3) size 8 mol/L LiCl (~20 ° C), vibrated and mixed, and placed at 4 ° C overnight. After centrifugation at 12000 × g for 20 min at 4 ° C, the supernatant was removed and the RNA precipitate remained untouched at the bottom of the tube. Take 3~5ml cold 2mol/L (4°C) LiCl, gently vibrate, centrifuge at 12000×g for 10min at 4°C, and remove the supernatant. Repeat 2~3 times of LiCl for 2~3 times until there is no pigment in the supernatant.
(4) Use 2 ml of 10 mmol/L. The precipitate was fully dissolved by Tris-HCl (pH 7.5), centrifuged at 12000 × g for 10 min at 4 ° C to remove the insoluble precipitate, and the supernatant was transferred to a 15 ml glass centrifuge tube to participate in 200 ul (1/10 size) of 2 mol/ LCH3COOH potassium (pH 5.5). Place in a 4 ° C warm bath for 30 min. Centrifuge at 12000 x g for 10 min at 4 °C. Transfer the supernatant to a new glass centrifuge tube, participate in 2.5 times the size of 100% alcohol, shake and place the RNA at -80 °C for 1~2h.
(5) Centrifuge at 9800 × g for 30 min at 4 ° C, remove the supernatant, and the RNA precipitate remained at the bottom of the tube. The pellet was washed with 1 to 2 ml of 70% cold alcohol (-20 ° C), centrifuged at 9800 x g for 5 min at 4 ° C, and the supernatant was removed. The RNA pellet was dried (20-30 min) at room temperature to the anhydrous beads in the tube.
(6) The precipitate was sufficiently dissolved with 200 ul of ultrapure water treated by dicarboxylic acid diethanol (DEPC). The solution was absolutely transferred to a 1.5 ml centrifuge tube, and the original tube wall and tube bottom were washed with 100 ul of ultrapure water treated by DEPC. The washing solution was combined in the RNA-dissolved precipitate to participate in a 1/10 size 3 mol/L CH3COOH sodium (pH 5). .2) and 100 times the size of 100% cold alcohol, mix and settle at -20 °C for 1 h. The mixture was centrifuged at 16000 × g for 30 min at 4 ° C, the supernatant was removed, and the RNA precipitated at the bottom of the tube was washed with 1 to 2 ml of 70% cold alcohol (-20 ° C), and centrifuged at 9800 × g for 5 min at 4 ° C. The supernatant was removed and the RNA precipitated to the bottom of the tube.
(7) RNA precipitation was dried at room temperature for 20 to 30 minutes. The RNA precipitate is dissolved in ultrapure water (100~300 ul) suitable for disposal by EDPC. The RNA solution can be retained for a long period of time at -80 °C or used for profiling.
(2) Participate in 20ml in a 50ml falcon tube to obtain a mitigation fluid. The group in which the liquid was taken out was changed to 0.2 mol/L sodium dehydrated sodium tetraborate (sodiumtetriboratedecahydrate), 30 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% (W/V) sodium dodecyl sulfate (SDS), 1%. Deoxycholate sodium salt. Further, 0.0309 g of dithiothreitol (DTT), 200 ul of ethyl phenyl polyglycol (NP-40) and 0.4 g of polyethylene pyrrolane ketone (PVP-40) were added and equilibrated at 80 ° C for 15 min. The samples were taken at -80 ° C, and after the thermal equilibration, the mitigation liquid was obtained and the vibration was mixed. Then participate in 800 ul of proteinase K (20 mg / ml), cover the tube cover, lay flat on a shaker with a speed of 120 r / min and a temperature of 42 ° C, and swing to obtain 1.5 h.
(3) After this, 1.6 ml of 2 mol/L KCl was added to the sample tube, and the mixture was shaken, and the sample tube was placed at 4 ° C for 1 to 1.5 hours. The extract solution was transferred to a glass centrifuge tube and centrifuged at 12000 x g for 20 min at 4 °C. The supernatant was placed in a new glass centrifuge tube and subjected to 1/4 (or 1/3) size 8 mol/L LiCl (~20 ° C), vibrated and mixed, and placed at 4 ° C overnight. After centrifugation at 12000 × g for 20 min at 4 ° C, the supernatant was removed and the RNA precipitate remained untouched at the bottom of the tube. Take 3~5ml cold 2mol/L (4°C) LiCl, gently vibrate, centrifuge at 12000×g for 10min at 4°C, and remove the supernatant. Repeat 2~3 times of LiCl for 2~3 times until there is no pigment in the supernatant.
(4) Use 2 ml of 10 mmol/L. The precipitate was fully dissolved by Tris-HCl (pH 7.5), centrifuged at 12000 × g for 10 min at 4 ° C to remove the insoluble precipitate, and the supernatant was transferred to a 15 ml glass centrifuge tube to participate in 200 ul (1/10 size) of 2 mol/ LCH3COOH potassium (pH 5.5). Place in a 4 ° C warm bath for 30 min. Centrifuge at 12000 x g for 10 min at 4 °C. Transfer the supernatant to a new glass centrifuge tube, participate in 2.5 times the size of 100% alcohol, shake and place the RNA at -80 °C for 1~2h.
(5) Centrifuge at 9800 × g for 30 min at 4 ° C, remove the supernatant, and the RNA precipitate remained at the bottom of the tube. The pellet was washed with 1 to 2 ml of 70% cold alcohol (-20 ° C), centrifuged at 9800 x g for 5 min at 4 ° C, and the supernatant was removed. The RNA pellet was dried (20-30 min) at room temperature to the anhydrous beads in the tube.
(6) The precipitate was sufficiently dissolved with 200 ul of ultrapure water treated by dicarboxylic acid diethanol (DEPC). The solution was absolutely transferred to a 1.5 ml centrifuge tube, and the original tube wall and tube bottom were washed with 100 ul of ultrapure water treated by DEPC. The washing solution was combined in the RNA-dissolved precipitate to participate in a 1/10 size 3 mol/L CH3COOH sodium (pH 5). .2) and 100 times the size of 100% cold alcohol, mix and settle at -20 °C for 1 h. The mixture was centrifuged at 16000 × g for 30 min at 4 ° C, the supernatant was removed, and the RNA precipitated at the bottom of the tube was washed with 1 to 2 ml of 70% cold alcohol (-20 ° C), and centrifuged at 9800 × g for 5 min at 4 ° C. The supernatant was removed and the RNA precipitated to the bottom of the tube.
(7) RNA precipitation was dried at room temperature for 20 to 30 minutes. The RNA precipitate is dissolved in ultrapure water (100~300 ul) suitable for disposal by EDPC. The RNA solution can be retained for a long period of time at -80 °C or used for profiling.
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