Large-scale preparation of Drosophila RNA
This method allows for the preparation of RNA from 100 Drosophila or approximately 2g of Drosophila embryos.
Operation method
Large-scale preparation of Drosophila RNA
Materials and Instruments
5mol LLiCl 70% Ethanol Phenol: chloroform (1:1) 20 mg ml Protease K 95% (V V) Ethanol .RNA homogenization buffer 3mol L Sodium acetate Move I. Materials and equipment Caveat 1) This method can be used to prepare RNA from Drosophila adults or embryos. for embryos, membranes must be removed and then neutralized, rinse membrane removed embryos with DEPC water before use.2) RNA precipitates may be difficult to dissolve after drying the plants under vacuum and can be heated for a few minutes and mixed gently with a pipette tip to aid in dissolution.3) A260/280should be greater than or equal to 1. 9. If the ratio is lower than 1.9, there may be protein contamination in the RNA preparation. The protein can be removed by phenol:chloroform extraction. For more product details, please visit Aladdin Scientific website.
1) 5mol/L LiCl
2)70% Ethanol: 70% (V/V) Ethanol, l0 mmol/L Tris-HCl (pH 7.5)
3) Phenol: chloroform (1:1): phenol equilibrated with 0.1 mol/L Tris-HCl (pH 8.0)
4) 20 mg/ml Protease K
5) 95% (V/V) ethanol
6)RNA homogenization buffer: 0.5%(m/V) SDS, 10 mmol/L EDTA\(PH8.0), 50 mmol/L NaCl, autoclaved, add Tris-HCl (PHI7.5) to a final concentration of 50 mmol/L
7) 3 mol/L sodium acetate ( PH5.5).
Operation method
1) Aspirate 2 ml of RNA homogenization buffer into a 7 ml Dounce homogenizer.
2) Add 37.5 Proteinase K and immediately place in Drosophila or its embryos. Homogenize evenly for 50 strokes with mortar and pestle type A.
3) Transfer homogenate to 15 mL conical tube. 37°C for 45-70 min.
4) Add equal volumes of phenol and chloroform, cover tightly and shake for 10s.
5) Separate the inorganic phase from the aqueous phase by centrifugation at 1000 g for 5 min.
6) Transfer the upper aqueous phase to a new tube.
7) Add 1 ml of new RNA homogenization buffer to the organic phase. Mix and then centrifuge as before to separate the layers.
8) Mix the aqueous phase twice and add 4 ml phenol:chloroform.
9) Centrifuge at 1000 g for 5 min to separate the organic and aqueous phases.
10) Transfer the upper aqueous phase to a new tube and add 0.5 ml of 3mol/L sodium acetate. After inverted mixing, add 2~2.5 times volume of 95% ethanol, inverted mixing.
11) Place at -20℃ for 2 h or -80℃ until solidification to precipitate DNA and RNA.
12) Centrifuge at 12000 g for lOmin, discard the supernatant. Rinse the precipitate with 5 ml of 70% ethanol and remove the supernatant.
13) Dry the precipitate in air or vacuum. Dissolve the precipitate with 0.5 ml of DEPC water. Dissolve the precipitate in 0.5 ml DEPC water and transfer to a 1.5 ml centrifuge tube.
14) To remove the DNA, add an equal volume of LiCl and mix with shaking.
15) Place at -20°C for 2 h or 4°C overnight to precipitate RNA.
16) Centrifuge at 12000 g for 10 min, remove supernatant and dry in air or vacuum.
17) Dissolve the precipitate in 0.4 ml of DPEC water. Can be left at 4°C overnight to dissolve
18) Add 50ul of 3mol/L sodium acetate and 0.9 ml of 95% ethanol. Mix upside down and place at -20°C for 2 h to precipitate RNA.
19) Centrifuge in a microcentrifuge at maximum speed for 10 min, remove the supernatant, and rinse the precipitate with 0.5 ml of 70% ethanol.
20) Dry the plants in air or vacuum and dissolve the precipitate in 180ul of DEPC water. Dissolve the precipitate in 180ul of DEPC water. Dispense into appropriate volumes and store at -70°C.