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Cloning a DNA segment from bacteriophage

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.

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Cloning a DNA segment from bacteriophage

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  1. 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?

  2. 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

  3. 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

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