Water for Northern and Southern Blotting
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.
More Information
- http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
- http://teachline.ls.huji.ac.il/72320/methods-tutorial/northern.html
- http://teachline.ls.huji.ac.il/72320/methods-tutorial/southern/southern-blot.html
- http://www.dnatube.com/video/1512/Southern-blot
- http://highered.mcgraw-hill.com/olc/dl/120078/bio_g.swf
- Hybridization and Southern Blots
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