Isolation of Total RNA (and Genomic DNA <70 Kb) from Cell Cultures and Tissues:

Proteinase K Procedure Using a Plasmid Kit


This procedure allows one to purify both total RNA and genomic DNA from the same tissue sample (NOTE 2). It is particularly useful when both types of nucleic acid are needed and the amount of tissue sample is limiting. (Please read the entire protocol before starting the prep.)


Special conditions for the type of NUCLEOBOND AX cartridge used

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. Homogenize the material directly 0.5 ml 2 ml 8 ml in the K1 lysis buffer (not included in the plasmid kit). Incubate at 50-60C for 1-2 hours preferably with gentle shaking.

2. Add 1/2 volume of cold buffer S3 250 Ál 1 ml 4 ml and mix gently. Pellet the SDS and debris by centrifugation at 4C and 12,000 x g for 10-20 minutes.

3. Transfer the supernatant to a 11 mg 45 mg 180 mg fresh tube, add 15 mg/ml KCl to a final concentration of 200 mM. (NOTE 4) Adjust the pH to 6.3 as required with dilute phosphoric acid.

4. Equilibrate a cartridge with 1 ml 3 ml 6 ml buffer N11 and load the sample.

5. Wash the cartridge with buffer N1 2 ml 4 ml 12 ml (NOTE 1)

6. Wash the cartridge with a 1:1 2 ml 4 ml 12 ml N1/N2 solution. (NOTE 1)

7. Elute the RNA with buffer N3. 1 ml 3 ml 6 ml

8. Add 0.8 volumes of iso-propanol 0.8 ml 2.4 ml 4.8 ml to the eluate. (NOTE 5) Incubate on ice for 15 minutes and centrifuge at 15,000 x g for 10-20 min. at 0C.

9. The RNA pellet is washed with 85% yes yes yes ethanol, air dried briefly,and dissolved in appropriate buffer for further handling or stored under 100% ethanol at -70C.

Optional for the recovery of genomic DNA. (NOTE 2) 10. Wash the cartridge with buffer N3. 3 x 1 ml 3 x 4 ml 3 x 12 ml (NOTE 3)

11. Elute the DNA with buffer N5. 1 ml 3 ml 6 ml

12. Add 0.8 volumes of iso-propanol 0.8 ml 2.4 ml 4.8 ml to the eluate. Incubate on ice for 15 minutes and centrifuge at 15,000 x g for 10-20 min. at 0C.

13. Wash the DNA pellet with 70% yes yes yes ethanol, air dry briefly,and dissolve in appropriate buffer for further handling.


NOTE 1: Please refer to Buffer Preparation for recommended N series buffer preparation procedure. Buffers comparable to N1 and 1:1 N1/N2 can also be made as follows: (1) for N1, substitute 50% N2 in 15% ethanol, pH 6.3 and make by mixing equal volumes of N2 and 15% ethanol, pH 6.3, for example, mix 5.0 ml of N2 and 5.0 ml of 15% ethanol, pH 6.3; (2) for 1:1 N1/N2, substitute 50% N2 from above for N1 and make by mixing equal volumes of N2 and 50% N2, for example, mix 5.0 ml of N2 and 5.0 ml of 50% N2.

NOTE 2: This procedure will only work well on the cartridges if one's genomic DNA fragments are not much larger than 70 Kb. (For most procedures requiring genomic DNA including PCR, restriction enzyme digestion and Southern blot analysis, lambda or cosmid library construction, 70 Kb fragments are more than sufficient.) If it is required that the majority of genomic DNA fragments be much larger, then Nucleobond AXG tubes, which contain loose resin in contrast to resin secured between membranes, should be used. Instructions on the use of AXG tubes can be found in steps 8 through 14 of the "Purification of Genomic DNA from Prokaryotes" procedure.

NOTE 3: Optional: When both RNA and DNA are being purified, an additional wash can be done with buffer N4 after the RNA is eluted, but before the DNA elution. This will remove any RNA that might still be bound, but could also elute some DNA. Alternatively, the eluted DNA could be treated with RNAse A, if required.

NOTE 4: Optional: When purifying total RNA and genomic DNA from plant samples, an extra step can be added at this point to reduce the level of polysaccharides in a prep prior to loading it onto a cartridge. This procedure is adapted from the one described by Micheals et al. (Michaels, S. D., M. C. John, and R. M. Amasino. 1994. Removal of Polysaccharides from Plant DNA by Ethanol Precipitation. BioTechniques 17:274-276.) and is performed as follows:

(a) Add 0.35 volumes of 100% ethanol to the solution in step 3 above prior to adjusting the pH.

(b) Mix and incubate on ice for 15-20 minutes. Centrifuge at 10,000 x g and 4C for 5-10 minutes.

(c) Titrate the pH of the supernatant to 6.3 and load it onto an equilibrated Nucleobond AX cartridge and continue from step 5 above.

NOTE 5: The ethanol, iso-propanol, and phosphoric acids available to most laboratories are DNAse/RNAse free. This can be easily checked for those used in your lab. Dilute the reagent approximately 10 to 1 in DEPC-treated water and add MgCl2 to a final concentration of 10 mM. Add 1-2 mg of RNA (or DNA), incubate at 37C for 30 minutes, and run on a 1.2% agarose gel (prepared with DEPC-treated water and RNAse-free loading and running buffers, if necessary) to determine whether the RNA (or DNA) was degraded. A control lane of RNA (or DNA) in DEPC-treated water alone will determine if any degradation resulted from the gel itself.


Buffer solutions                                        Storage
K1- 50 mM Tris-HCl, pH 8.0, 50 mM KCl , 10-20 mM EDTA,                4C
1 mM dithiothreitol, 0.5% SDS, 200 mg/ml proteinase K
S3 - 2.8 M K-Acetate, pH 5.2                                          4C
N1 - 100 mM Tris, 15% ethanol and 400 mM KCl adjusted                 RT
with phosphoric acid to pH 6.3
N2 - 100 mM Tris, 15% ethanol and 900 mM KCl adjusted                 RT
with phosphoric acid to pH 6.3
N3- 100 mM Tris, 15% ethanol and 1150 mM KCl adjusted                 RT
with phosphoric acid to pH 6.3
N4- 100 mM Tris, 15% ethanol and 1300 mM KCl adjusted                 RT
with phosphoric acid to pH 6.3
N5- 100 mM Tris, 15% ethanol and 1000 mM KCl adjusted                 RT
with phosphoric acid to pH 8.5 

To make your own buffers or for more information please refer to Buffer Preparation and Nucleobond AX Buffers.

Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

Back to the Nucleobond AX applications guide


Updated 1/20/98