Transcript BamHI
Part 1. Gel electrophoresis • DNA is negatively charged (because of phosphate backbone) • DNA will be attracted to positively charged poles and repelled from negatively charged ones Part 2. Restriction Endonucleases (aka restriction enzymes) • Enzymes that “cut” DNA in a sequencespecific manner • Serve as a natural defense mechanism for bacteria against viral infection • Bacteria protect their DNA from cutting by their own enzymes through methylation Examples Enzyme • EcoRI • HindIII • BamHI • EcoRV Recognition sequence GAATTC AAGCTT GGATCC GATATC • Recognition sequences are usually 4-8 base pairs in length and are usually palindromic A closer look…. BamHI BamHI 5’….ACTGTACGGATCCGCTA….3’ 3’….TGACATGCCTAGGCGAT….5’ A closer look…. BamHI 5’….ACTGTACG GATCCGCTA….3’ 3’….TGACATGCCTAG GCGAT….5’ Ligations • When DNA molecules with sticky ends come together, only hydrogen bonds between complimentary nucleotides are reformed • These H-bonds are not stable enough to be permanent • DNA ligase=enzyme that joins the ends of DNA and re-establishes the phosphodiester bond in the DNA molecule DNA Ligase 5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’ Running a gel • Molten agarose is poured into a casting tray and a comb is placed • After the agarose solidifies, the comb is removed leaving wells where the DNA will be loaded • DNA samples are mixed with tracking dye which contains sucrose (to weigh down the DNA) and dyes so that you can visualize migration • A buffer containing ions (to conduct an electric current) is placed in the chamber around the gel Gel electrophoresis - electrode DNA fragments + electrode Agarose gel ~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ Gel electrophoresis - electrode + electrode current ~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ Movement of DNA fragments in agarose gels • There is a linear relationship between the migration rate of a given DNA fragment and the logarithm of its size (in basepairs). • Larger molecules move more slowly through the gel because of more friction An ethidium-stained gel photographed under UV light **Each band that you see is a collection of millions of DNA molecules, all of the same length!!