Guanidine hydrochloride rapidly inhibits RNAase activity while lysing cells, and this method utilizes this feature to isolate Drosophila RNA.
Operation method
Small-scale rapid preparation of Drosophila RNA
Materials and Instruments
Northern Sample buffer lmol L Acetic acid Phenol Chloroform DEPC treated water GHCL solution Anhydrous ethanol Move I. Materials and equipment Caveat 1) If very pure RNA is required, precipitate the RNA three times as described in steps 6) through 9).2) To minimize the effect of the RNAase, gloves should be worn and changed frequently, and all solutions should be prepared in DEPC-treated water. For more product details, please visit Aladdin Scientific website.
1) Northern sample buffer: 2.2 mol/L formaldehyde, 1 mol/LMOPS, 50% formamide
2)lmol/L acetic acid
3) Phenol: Chloroform (1:1)
4)DEPC treated water
5) GHCL solution: 5 mol/LDTT, 7.5 mol/L guanidine hydrochloride, 25 mmol/L sodium acetate (pH 7.0)
0.5% N-lauroyl sarcosine
6) Anhydrous ethanol
II Methods of operation
1) Put Drosophila or Drosophila embryos into a 1.5 ml centrifuge tube with GHCL solution and plant 50ul of GHCL solution for each Drosophila or 50-100 Drosophila embryos, and treat up to 20 Drosophila.
2) Rapid homogenization of the sample with a small mortar and pestle.
3) Add equal volume of phenol: chloroform to the homogenate and mix by shaking.
.
4) Centrifuge in a microcentrifuge at maximum speed for 2 min.
5) transfer the upper aqueous phase to a new small centrifuge tube
6) Add lmol/L acetic acid and 25ul of anhydrous ethanol per 50ul of GHCL solution to precipitate the RNA.
7) Place at -20℃ for 3~24 h.
8) Centrifuge in a microcentrifuge at maximum speed for 5 min.
9) Carefully remove the supernatant and dissolve the precipitate in half the volume of GHCL solution used initially in a minimum volume of (50ul), and then add the supernatant to the solution.
10) Reprecipitate the RNA as in step 9).
11) If necessary, precipitate the RNA again as described above.
12) Add the volume of GHCL solution used in step 1 to the RNA precipitate in anhydrous ethanol and mix.
13) Centrifuge in a microcentrifuge for 15 mim at maximum speed.
14) Remove the ethanol and wash again with anhydrous ethanol if necessary.
15) The RNA precipitate can be stored in a suitable buffer or in anhydrous ethanol plants. If the RNA is to be used for Northern blot analysis, it is best to dissolve it in Northern Sample Buffer.