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Water for Northern and Southern Blotting

 
Purificazione dell'acqua
 
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Impact of Water

Ultrapure water is used for many steps of blotting: sample, buffer, gels and rinsing solutions preparation. Prior to blotting itself, either for Southern or Northern blotting, the sample preparation implies a particular care of water quality used to protect raw material (respectively DNA or RNA).

The major sample preparation steps are the isolation by extraction, amplification and a pretreatment to denature RNA to prevent formation of base-paired secondary structure.

Water Quality Parameters

  • Ions
    Charged molecules can interfere during migration electrophoresis steps by changing the global charge of the solutions (buffer, sample…) and then either accelerating or delaying the migration.

    The concentration of ions is also important during the hybridization step. In order to carry on the hybridization in optimum conditions, specific concentrations should be respected. Starting with water virtually free of ions (resistivity 18.2 MΩ•cm @ 25°C) ensures the preparation of the buffers at the right ionic strength and pH. 
  • Organics
    Organic molecules can create interferences in the hybridization process. Those molecules that are negatively charged can behave just as the DNA and RNA and bind non-specifically in place of the DNA or RNA. Positively charged molecules can actually bind to the DNA or RNA and the oligonucleotides, retarding or interfering with the hybridization process. Consequently, low TOC value (< 10 ppb or µg/L) is recommended to ensure good water quality in terms of organic content. 
  • Nucleases
    Nucleases degrade the NA samples. These enzymes are efficiently removed using ultrafiltration. The ultrafilter is installed at the outlet of the purification unit, just before water is drawn from the system and used. Please refer to the section on nuclease-free water. 
  • Bacteria
    Bacteria liberate nucleases that degrade Nucleic Acids samples. They contain DNA, which can interfere with DNA to be checked. Finally, they release ions and organics that can have a negative effect on blotting steps Water purified with an Ultrafilter (BioPak) has bacteria levels < 0.1 cfu/mL. 
In summary, considering the overall processes associated with the blotting steps, water should be nuclease-free and bacteria-free, have a high resistivity and a low TOC. All this can be obtained using an ultrafiltration device at the point-of-use (the outlet) of a water purification unit delivering high purity water. Please refer to the following sections for more information: Nuclease-free water, gel electrophoresis, PCR and DNA Sequencing.

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