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Labs 10 to 12

Labs 10 to 12. Southern Blotting & Physical Mapping. 3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology. Gel Electrophoresis and Gene Mapping Map of the gene Determine which restriction enzymes cut the gene and pinpoint the exact location of the sites

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Labs 10 to 12

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  1. Labs 10 to 12 Southern Blotting & Physical Mapping

  2. 3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology • Gel Electrophoresis and Gene Mapping • Map of the gene • Determine which restriction enzymes cut the gene and pinpoint the exact location of the sites • Restriction map is a map of known restriction sites within a sequence of DNA using restriction enzymes

  3. Gel Electrophoresis and Mapping Gene Structure with Restriction Enzymes Restriction Map -Physical map of the gene is created to determine which restriction enzymes cut the cloned gene. Procedure:Cut with different restriction enzymes and characterize with gel electrophoresis.

  4. Gel Electrophoresis and Mapping Gene Structure with Restriction Enzymes Uncut DNA: Lane 1: 7 kb BamHI, Lane 2: 4.5 kb, 2.5 kb PstI, Lane 3: 5.5 kb, 1.5 kb Bam HI + PstI, Lane 4: 3.0 kb, 2.5 kb, 1.5 kb DNA pieces are negatively charged and separate by size with the smallest pieces running the fastest

  5. Chromosome Location and Copy Number • Southern blotting • Digest chromosomal and plasmid DNA with restriction enzymes • Fragments are separated by agarose gel electrophoresis • Gel is treated with alkaline solution to denature the DNA • Fragments are transferred onto a nylon or nitrocellulose filter (called blotting) • Filter (blot) is incubated with a probe and developed • Number of bands on film represents gene copy number • A hybridization probe is a small fragment of DNA or RNA which is used to detect in DNA or RNA samples the presence of nucleotide sequences that are complementary to the sequence in the probe

  6. Restriction fragment analysis by Southern blotting Development

  7. Procedure • We will perform a Southern blot analysis of the pDsRed2 plasmid, which harbors the DsRed gene (for red fluorescence), using a non-radioactive probe complementary to the beginning of the gene • 3 Steps (days): • Gel electrophoresis, denaturation and transfer (Southern) • Hybridization • Probe detection Lab Manual: Pg. 99-106 (Day 1) 106-107 (Day 2) 108-109 (Day 3) http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio_g.swf::Southern%20Blot

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