The transfer of macromolecules such as nucleic acids and proteins to solid-phase membranous support is known as blotting. Fragments of DNA and RNA molecules separated by gel electrophoresis are transferred to a nylon or nitrocellulose membrane in a process termed as Southern and Northern blotting, respectively. Southern blotting was introduced by Edwin Southern in 1975 as a method to detect specific sequences of DNA in DNA samples. The other blotting techniques emerged from this method have been termed as Northern (for RNA), Western (for proteins), Eastern (for post-translational protein modifications) and Southwestern (for DNA-protein interactions) blotting.
Southern and Northern blotting protocols involve the following major steps:
Figure 1.Steps involved in DNA/RNA blotting procedure
*PerfectHyb™ Plus Hybridization Buffer (H7033) can be used in place of Denhardt, prehybridization, and hybridization solutions. PerfectHyb™ Buffer has been optimized to yield maximum signal with minimum background in hybridizations as short as 1-2 hours. PerfectHyb™ Plus has been formulated to work in any hybridization protocol, utilizing any type of probe, and on any type of membrane (positively charged or neutral nylon and nitrocellulose).
Sigma-Aldrich offers GenElute™ kits for isolation of DNA from plants and fungi (E5038), mammalian cells or tissue (G1N70, G1N10 and G1N350) and blood (NA2010 and NA2020). The detailed protocol of DNA isolation by using GenElute™ kits may be found here.
Additionally, DNAstable® kits (93000-001-1EA, 93021-001-1EA, 53091-016-2ML and 93121-017-1EA) may be used in case the DNA is being shipped or for storage and stabilization of DNA at room temperature.
Digest the DNA sample with appropriate restriction enzyme for 2-24 h at 37 °C. If the DNA sample is clonally derived a digestion time of 1-2 h is sufficient. For genomic DNA, overnight incubation is generally required with excess enzyme (5-10X).
If necessary, concentrate the digested DNA using ethanol precipitation. The traces of ethanol must be completely removed before separation on the gel.
Resolve the digested DNA on agarose gel. TAE should be used for shorter runs (<4hrs) and larger DNA fragments, while TBE should be used for longer runs (>4hrs) and smaller DNA fragments (<1kb). Alternatively, use Bionic™ Buffer (B6185) for sharper band resolution in less time than TAE or TBE (for more information see Bionic™ Buffer Data). Stain the gel with ethidium bromide and acquire the gel image using UV transilluminator. If ethidium bromide has been incorporated into the agarose prior to electrophoresis, the gel image can be acquired immediately after the run.
During the transfer step the DNA (or RNA) from the electrophoresis gel will be transferred onto a nylon membrane so it may be accessible to a probe for hybridization and detection.
Figure 2.Southern/northern blot transfer assembly
The prehybridization step (also known as blocking) is done to minimize non-specific attachments of a probe to the nylon membrane. Salmon sperm DNA is commonly used as a blocking agent to prevent the probe from sticking to the membrane, ensuring that it will only interact with the desired DNA bands that have been transferred to the membrane. Prepare the prehybridization solution as follows or use PerfectHyb™ Plus buffer (H7033):
A complementary strand of DNA (a probe) is used to detect the desired sequence that should be present on the membrane. DNA from two different sources combine to make a "hybrid" dsDNA, but only if the two strands are homologous.
The protocol for Northern blotting is similar to that of Southern blotting. However, RNA sequences are separated on denaturing agarose gel incorporated with formaldehyde.
If ethidium bromide has been incorporated into the agarose prior to electrophoresis, the gel image can be acquired immediately after the run.
The transfer setup, prehybridization, hybridization, and detection protocols are the same as those for Southern blotting.
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