Part 1. Gel electrophoresis

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Presentation transcript:

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

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

Agarose Gel Tray. Gel trays differ depending on the manufacturer Agarose Gel Tray. Gel trays differ depending on the manufacturer. Each has some method of sealing the ends so that liquid agarose can mold into a gel. Some gel trays, such as those made by Owl Separation Systems, make a seal with the box, so casting a gel is simple. Other trays require masking tape on the ends to make a mold. Still others, like the one shown here, have gates that screw into position: up for pouring the gel and down for running the gel. Molecules in a Gel Box. If negatively charged molecules are loaded into the wells and run on the gel, the smaller ones run faster and farther than the larger ones toward the positive electrode. This is because smaller molecules pass more easily through the tiny spaces of the gel network.

Gel electrophoresis Agarose gel - electrode + 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

Visualizing DNA Ethidium bromide A fluorescent dye visualized when excited by UV light Intercalates into the DNA molecule, thereby “staining” it Gel is soaked in a solution of EtBr and the DNA bands take up the dye Then the gel is placed under UV light and visualized and/or photographed

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!!

Part 2. Restriction Endonucleases (aka restriction enzymes) Enzymes that “cut” DNA in a sequence-specific 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 Recognition sequence EcoRI GAATTC HindIII AAGCTT BamHI GGATCC EcoRV GATATC Recognition sequences are usually 4-8 base pairs in length and are usually palindromic

A closer look…. BamHI 5’….ACTGTACGGATCCGCTA….3’ 3’….TGACATGCCTAGGCGAT….5’

A closer look…. BamHI 5’….ACTGTACGGATCCGCTA….3’ 3’….TGACATGCCTAGGCGAT….5’

A closer look…. BamHI 5’….ACTGTACGGATCCGCTA….3’ 3’….TGACATGCCTAGGCGAT….5’

A closer look…. BamHI 5’….ACTGTACG GATCCGCTA….3’ 3’….TGACATGCCTAG GCGAT….5’

A closer look…. BamHI GATCCGCTA….3’ GCGAT….5’ 5’….ACTGTACG 3’….TGACATGCCTAG “sticky ends” -can bind with other DNA molecules with the same overhangs

A closer look…. EcoRV 5’….ACTGTACGATATCGCTA….3’ 3’….TGACATGCTATAGCGAT….5’

A closer look…. EcoRV 5’….ACTGTACGAT ATCGCTA….3’ 3’….TGACATGCTA TAGCGAT….5’

A closer look…. EcoRV 5’….ACTGTACGAT ATCGCTA….3’ 3’….TGACATGCTA TAGCGAT….5’ “blunt ends” -can bind with other DNA molecules with blunt ends

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’

DNA Ligase 5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’

DNA Ligase 5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’

Rate of DNA Movement though a Gell DNA moves through the gel at a rate that is inversely proportional to the log10 to the molecular weight Chart the distance migrated vs. molecular weight of the Lambda-HindIII fragments on semilog paper. Draw a line through the data points Then use this line to calculate the size of the BamHI and EcoRI digest bands

base pairs Distance migrated

x bp base pairs Distance migrated