Notes March 3rd

Notes March 3rd - Notes March 3rd Fig 24-29 - Southern...

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Notes March 3 rd Fig 24-29 - Southern blotting - Method for identifying a particular DNA fragment as long as igf you have a radioactive probe to hybridize it - Takes advantage of the ability of nitrocellulose to bind to single stranded DNA but not duplex - Following el electrophoresis of dsDNA, gel is soaked in NaOH to convert DNA to its single stranded form - Gel is then overlaid by a sheet of nitrocellulose paper - Molecules in the gel are forced through the nitrocellulose by drawing out the liquid with a stack of absorbent towels compressed against the far side of the nitrocellulose, or by using an electrophoretic process (electroblotting) - Single stranded DNA binds to the nitrocellulose at the same position it had in the gel; after drying at 80 degrees, which permanently fixes the DNA in place, the nitrocellulose sheet is moistened with a minimal quantity of solution containing 32 P-labeled single stranded DNA or RNA prove that is complementary in sequence to the DNA of interest; - Moistened nitrocellulose is heald at a suitable renaturing temperature for several hours to permit the probe to hybridize to its target seuqence - then washed to remove the unbound radioactive probe, dried, and autoradiographed by placing it for a time over a sheet of x-ray film; the positions of the molecules that are complementary to the radioactive probe are indicated by a blackening of the developed film - Done on filter paper - Gel is put into alkali to denature the DNA to single stranded DNA - Nitrocellulose binds ssDNA with high affinity o If you touch the gel with the nitrocellulose and blot it, the DNA will get bound to the
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This note was uploaded on 09/14/2011 for the course BIO 362 taught by Professor Walikarzai during the Spring '10 term at SUNY Stony Brook.

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Notes March 3rd - Notes March 3rd Fig 24-29 - Southern...

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