Southern Blotting Lab Report
Southern Blotting
Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome.
The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.
This diagram shows the basic steps involved in a Southern blot.
southern blotting
Let's look at this technique in greater detail.
1. Digest the DNA with an appropriate restriction enzyme. …show more content…
In this procedure, a vacuum sucks SSC through the membrane. This works similarly to capillary action, except more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)
After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)
5. Probe the membrane with labeled ssDNA. This is also known as hybridization. Whatever you call it, this process relies on the ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands. Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.
A prehybridization step is required before hybridization to block non-specific sites, since you don't want your single-stranded probe binding just anywhere on the membrane.
To hybridize, use the same buffer as for prehybridization, but add your specific
Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome.
The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.
This diagram shows the basic steps involved in a Southern blot.
southern blotting
Let's look at this technique in greater detail.
1. Digest the DNA with an appropriate restriction enzyme. …show more content…
In this procedure, a vacuum sucks SSC through the membrane. This works similarly to capillary action, except more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)
After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)
5. Probe the membrane with labeled ssDNA. This is also known as hybridization. Whatever you call it, this process relies on the ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands. Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.
A prehybridization step is required before hybridization to block non-specific sites, since you don't want your single-stranded probe binding just anywhere on the membrane.
To hybridize, use the same buffer as for prehybridization, but add your specific