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LAB4.NEW - Recombinant DNA Session 04 Southern blotting...

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Recombinant DNA Session 04: Southern blotting BACKGROUND Blots! North, South, East (not yet), and West! Blots are an analytical technique to detect the presence and state of nucleic acid and protein sequences. The transfer of DNA from an agarose gel to a membrane sheet is called Southern blotting, after its inventor, Ed Southern. The transfer process uses buffer flow by capillary action to carry the DNA out of the gel and onto the membrane. It is also possible to use vacuum filtration or electrophoretic transfer to move the DNA out of the gel. Advantages of the transfer process are: 1. It is not possible to immobilize the DNA in the gel whereas the transferred DNA binds very tightly to the nylon once it is baked on. 2. The "target" DNA would not be readily accessible to the hybridization probe in the gel whereas the nylon membrane is thinner and more porous, allowing the probe to readily find its target. The Membrane Nylon offers the advantages of durability and the ability to be reprobed without losing the target DNA. It binds DNA fragments less than 500 bp much more efficiently than nitrocellulose does. The capacity of charged nylon to bind DNA is 4 or 5 times higher than for nitrocellulose. The disadvantages of nylon are that it can have a higher background of nonspecific binding of the probe. Nylon also tends to be more expensive. It should be handle with gloved hands or forceps, preferably by edges. Grease from fingerprints will coat surface, preventing DNA transfer. Never rest nylon directly on the lab bench. Whatman 3MM paper (also expensive!) will provide a wick or a reservoir of SSC buffer to transfer DNA out of the gel and onto the nylon. Melting of DNA sequences The principal forces holding the DNA double helix together are hydrogen bonds between bases in opposite strands and hydrophobic interactions between bases within the same and opposite strands. Phosphate groups tend to destabilize ds(double stranded)DNA. The melting temperature of DNA depends on its base composition. G-C base pairs in double-stranded DNA can form 3 hydrogen bonds as opposed to 2 hydrogen bonds for A-T base pairs. There is a linear increasing relationship between the percent G-C content and the melting temperature of DNA. The negative charges of phosphate groups on phosphate-sugar backbone cause electrostatic repulsion and tend to blow dsDNA apart. A high cation concentration stabilizes double-stranded DNA by shielding the phosphate groups from each other and raises the melting temperature. The length of the sequence being melted influences the melting temperature. Short sequences, because there are fewer forces to hold them together, are less stable. GOALS 23
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To melt the ds DNA into ss DNA fragments. To transfer the ss DNA fragments from the agarose gel to more permanent nylon membrane.
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