Northern blotting is a method used in molecular biology to identify specific ribonucleic acid (RNA) fragments which have been resolved by gel electrophoresis via transfer of the band pattern to a filter matrix. A recent change to the Northern blot procedure has eliminated the generation of mixed waste (waste that contains chemicals regulated by the Resource Conservation and Recovery Act combined with radioactive materials) by substituting either methanol or ethanol with Sodium Chloride/Sodium Citrate (SSC) and Sodium Dodecylsulfate (SDS) during the blot washing.
Applicable Regulations
10 CFR 20 Subpart K.

40 CFR Parts 260-268.

State of Michigan Act 451 Part 111.

Overview of Procedure
The Northern blot procedure transfers RNA separated from electrophoresis gel to a filter matrix for subsequent detection and analytical techniques. Typically the Northern blot procedure generates methanol or ethanol waste mixed with phosphorus-32 (32P) during the blot washing. A recent change to the procedure has eliminated the need for either methanol or ethanol and has replaced them with Sodium Chloride/Sodium Citrate (SSC) and Sodium Dodecylsulfate (SDS). When SSC & SDS are used during the wash it eliminates the generation of mixed waste from the procedure. The wash will still contain 32P that has been used to radiolabel the nucleotides and will be managed for disposal as low-level radioactive waste (LLRW). The new procedure is detailed below.

Waste Minimization Procedure
Northern Blot Analysis Procedure

Set up Agarose gel with radiolabeled sample for electrophoresis. This will separate molecules by size for further work.

  1. Run gel at 80 volts for 3-4 hours.
  2. Photograph gel under UV light box.
  3. Cut gel and soak for 30 minutes in NaOH and NaCl mixture followed by Tris buffer and NaCl.
  4. Soak Hybond-N filter for 20 minutes in H2O and 10 minutes in SSC.
  5. Transfer filter, then perform UV cross linking by placing wet membrane, RNA side up, on a glass plate for irradiation in the Stratalinker.
  6. Perform 3 separate washes with different specific concentrations of SSC and SDS to improve retention on the membrane and remove impurities.

The membrane is now ready for further detection and analytical techniques.

Known Limitations
None known.

Safety & Health Precautions/Personal Protective Equipment
Follow all applicable safety and health protocols and regulations as established by your institution.

The results of the new procedure are comparable to those of the old procedure; the sensitivity is equivalent to the previously used method. The time necessary to complete the blot washing using the new procedure has not changed.

Another benefit to using the new procedure is the associated cost savings to the U-M. The cost of processing and disposal of aqueous LLRW is much less than that of mixed waste.

None known.

Project Related Costs
The total costs from pickup to disposal of the different types of LLRW are as follows:

1. Liquid LLRW (non-mixed) $58 per gallon.

2. Liquid LLRW (mixed) $158 per gallon.

Therefore, the U-M's total saving is $100 for each gallon of mixed waste not generated by using the new blot washing procedure.