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This lab report outlines the cloning of a DNA segment from bacteriophage using recombinant plasmid DNA purified from bacterial cells. We performed restriction digests with EcoRI and BamHI to isolate specific DNA fragments. Following the digests, agarose gel electrophoresis was conducted to analyze the results and verify the sizes of the restriction fragments, using restriction digest maps for reference. The integrity and efficiency of the cloning process were assessed through comparison of fragment sizes with expected values from the restriction maps.
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Cloning a DNA segment from bacteriophage Recombinant plasmid DNA purified from bacterial cells Started restriction digests last week Why digesting with EcoRI again? Why digesting with EcoRI + BamHI?
Cloning a DNA segment from bacteriophage Recombinant plasmid DNA purified from bacterial cells LAB: Pour agarose gel Retrieve samples Add 5 µL of sample buffer (dye) - contains RNase Load 15 µL of each digest onto agarose gel Examine results - use restriction digest maps for EcoRI and BamHI
Cloning a DNA segment from bacteriophage Restriction map of lambda DNA - EcoRI Sizes of restriction fragments 21,226 4878 5643 7421 5804 3530 21,226 26,104 31,747 39,168 44,972 Position of EcoRI restriction sites Restriction map of lambda DNA - BamHI Sizes of restriction fragments 5505 16841 5626 6527 7233 6770 5505 22346 27972 34499 41732 Position of BamHI restriction sites
