Archive image from page 48 of The development of a cosmid. The development of a cosmid map of chromosome 12p13 . developmentofcos00belk Year: 1998 Figure 12. PCR of Cosmid 170G6 with Forward and Reverse 950 Primers. A positive band for STS marker D12S950 is observed. Lane 1 is empty, Lanes 2 and 6 contain 1 KB ladder. Lane 3 contains Total Human DNA (K562), Lane 4 contains Chromosome 12, and Lane 5 contains cosmid 170G6. RESTRICTION Enzyme DIGESTIONS. The use of restriction endonucleases is one way to show the relationships between different pieces of recombinant DNA. Restriction endon
Archive image from page 48 of The development of a cosmid. The development of a cosmid map of chromosome 12p13 . developmentofcos00belk Year: 1998 Figure 12. PCR of Cosmid 170G6 with Forward and Reverse 950 Primers. A positive band for STS marker D12S950 is observed. Lane 1 is empty, Lanes 2 and 6 contain 1 KB ladder. Lane 3 contains Total Human DNA (K562), Lane 4 contains Chromosome 12, and Lane 5 contains cosmid 170G6. RESTRICTION Enzyme DIGESTIONS. The use of restriction endonucleases is one way to show the relationships between different pieces of recombinant DNA. Restriction endonucleases recognize a specific four to eight base sequence within double-stranded DNA and cleave both strands, cutting approximately every 256 bases for a four base recognition site and every 4096 bases for a six base recognition site. Of the three types of endonucleases the Type II restriction enzymes are the most useful for DNA manipulation because they cleave at sites within their recognition sequence whereas Types I and III cleave at sites distant from the recognition site. In nature, bacteria use restriction endonucleases for protection from viral infection. In the laboratory, restriction enzymes can be used to characterize DNA. Restriction maps show relative positions of restriction cleavage sites within a DNA molecule and can provide a framework for locating base sequences within a fragment of DNA. Treatment of several different DNAs with the same restriction enzymes produces a series of fragments of defmed sizes. If these fragmented DNA molecules are run on gel electrophoresis the identically sized regions within the DNAs are easily identified. If two pieces of DNA share a portion of the same sequence they will be fragmented in equal sized pieces and these are visualized through electrophoresis (FIGURES 13, 14). 23
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