What is the use of NaOH in Southern blotting?
It uses the flow of a 0.4M NaOH solution to deposit the DNA fragments on the membrane. A wet sheet of blotting paper acts as a wick for the transfer solution, which is drawn up by a stack of dry paper through the gel/membrane sandwich.
Why is the gel treated with NaOH before blotting?
If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA.
Which transfer membrane is used for Southern blotting?
nitrocellulose membrane
This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane.
What is the principle of Southern blotting technique?
Principle. Southern blotting is based on the principle of separation of DNA fragments by gel electrophoresis followed by the identification by labeled probe hybridization. The DNA fragments are separated based on their size and charge during electrophoresis.
Which probes are most commonly used in Southern blotting?
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.
What do probes attach to?
Probes are stretches of DNA or RNA that we’ve attached a label to. The label allows us to see where the DNA binds either in a cell, or in a chromosome, or even in pure isolated DNA. We label probes with different molecules to follow them.
What is Southern blot used for?
A Southern blot is a laboratory method used to detect specific DNA molecules from among a many other DNA molecules.
What is the Southern blotting procedure?
Southern blotting is a laboratory technique used to detect a specific DNA sequence in a blood or tissue sample. The membrane is exposed to a DNA probe labeled with a radioactive or chemical tag. If the probe binds to the membrane, then the probe sequence is present in the sample.
Why is Southern blotting needed when genomic DNA is used?
Genomic DNA, containing the gene to be cloned, is blotted and hybridized to identify one or more restriction fragments containing the desired gene, and Southern blotting is used later in the project when a tentative clone has been isolated, to verify that the clone does indeed contain the desired gene and possibly to …
What is Southern blot test used for?
​Southern Blot Southern blotting is a laboratory technique used to detect a specific DNA sequence in a blood or tissue sample. A restriction enzyme is used to cut a sample of DNA into fragments that are separated using gel electrophoresis. The DNA fragments are transferred out of the gel to the surface of a membrane.
What are the tools and equipment needed to do the Southern blotting technique?
Materials Required
- Reagents for DNA isolation and purification.
- Reagents for restriction digestion of DNA.
- Reagents and buffers for agarose gel electrophoresis.
- Apparatus for agarose gel electrophoresis.
- Whatman filter papers.
- Paper towels.
- Positively charged nylon membrane.
- Salmon sperm DNA.
How is the Southern blot used in DNA analysis?
Southern blot is the process of transfer of DNA fragments that are separated by electrophoresis onto a membrane for immobilization and identification. Southern blotting has been adopted as a routine procedure for the analysis of DNA samples for different applications.
Where does the name Southern blotting come from?
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.
How does a vacuum work in Southern blotting?
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.
When do you soak DNA in NaOH for depurination?
Denature the DNA (usually while it is still on the gel). For example, soak it in about 0.5M NaOH, which would separate double-stranded DNA into single-stranded DNA. Only ssDNA can transfer. A depurination step is optional.