For the isolation of total RNA, rRNA, viral RNA or tRNA from crude extracts containing proteins, polysaccharides and DNA, the extract may be cleared by a centrifugation step, precipitated and redissolved in buffer N0 to get a low viscosity solution. Alternatively, a sample preparation may be accomplished by phenol extraction. The aqueous phase of the phenol/chloroform-extraction step is precipitated with iso-propanol. The crude RNA pellet is redissolved in buffer R0.
RNA containing reaction mixtures are adjusted to binding conditions by adding 1/5 volume of buffer R3 and adjusting the pH to 6.3 with dilute phosphoric acid following which the sample is loaded onto the cartridge. Such reaction mixtures may contain radioactive labeled RNA probes or "run off" cRNA transcripts.
For good binding conditions for different RNA's on NUCLEOBOND AX use pH 6.3 and a final salt concentration of 0.2M.
Step of the procedure AX 20 AX 100 AX 500 Amount of cells/mass of tissue used 4 x 10^4 2 x10^5 1 x 10^6 /20 mg /100 mg /0.5 g 1. Buffer W1 is added. 0.5 ml 2 ml 8 ml
Special conditions for the type of NUCLEOBOND AX cartridge used
2. Add W2 to the solution and 0.5 Ál 2 Ál 10 Ál homogenize with a commercial homogenizer (e.g. Polytron 4 x 20 sec). Other tools for homogenization tools may be used alternatively.
3. Add solution W3, mix the sample, 50 Ál 200 Ál 800 Ál and incubate for 15 min.at 0C.
4. Add buffer W4, mix carefully, and 500 Ál 2 ml 8 ml incubate for 15 minutes at 0C.
5. Centrifuge the mixture at 12,000 yes yes yes x g for 35 minutes at 0C.
6. Add iso-propanol to the 850 Ál 3.4 ml 13.5 ml supernatant, mix carefully, and incubate for 10 minutes at 0C.
7. Repeat step 5 yes yes yes
8. Discard the supernatant and 3 ml 7.5 ml 15 ml dissolve the pellet in buffer W5 very carefully.
9. If necessary, remove any particulate matter by centrifugation (15,000 x g, 20 min., 0C) and collect the supernatant.
10. Add buffer W6 to this cellular 1 ml 2.5 ml 5 ml extract (final pH of about 6.3 should be checked).
11. Equilibrate a NUCLEOBOND AX 1 ml 3 ml 6 ml cartridge with buffer R1.
12. Transfer the pretreated sample yes yes yes onto the cartridge.
13. Wash away contaminating 2 x 2 ml 2 x 4 ml 2 x 12 ml substances using the buffers given below for the purification of the following RNA's:
16. The RNA pellet is washed with yes yes yes 85% ethanol, dried for some minutes and dissolved in appropriate buffer for further handling or stored under 100% ethanol at -70C.
Compound Buffer R - 100 mM Tris-Acetate, 15% Ethanol, pH 6.3 + the following KCl salt concentrations
Elution conditions for different RNA's
tRNA 0.45 - 0.65 M rRNA 0.95 - 1.10 M mRNA 0.70 - 1.15 M 5S RNA 0.65 - 0.85 M total RNA 0.45 - 1.15 M
Buffer solutions Storage W1 - 50 mM Tris-Acetate, 4 M Guanidine thiocyanate, 10 mM MgCl2, pH 7.8 4C W2 - b-Mercaptoethanol 4C W3 - 20% Triton X 100 RT W4 - 3 M Sodium Acetate, pH 6.5 RT W5 - 10 mM Tris-Acetate, 1 mM EDTA pH 7.8 RT W6 - 200 mM Tris-Phosphate, 1.5 M KCl, pH 6.3 RT R1 - 100 mM Tris-Acetate, 15% Ethanol, 400 mM KCl, pH 6.3 RT R2 - 100 mM Tris-Acetate, 15% Ethanol, 900 mM KCl, pH 6.3 RT R3 - 100 mM Tris-Acetate, 15% Ethanol, 1150 mM KCl, pH 6.3 RT
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
Back to the Nucleobond AX applications guide