1. 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

2. 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

3. 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.

4. Gel electrophoresis Agarose gel - electrode + electrode
DNA fragments
+ electrode
Agarose gel
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buffer
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5. Gel electrophoresis - electrode + electrode current
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buffer
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7. 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

8. 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

9. 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!!

10. 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

11. 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

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

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

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

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

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

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

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

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

20. 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

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

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

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

24. 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

25. base pairs
Distance migrated

26. x bp
base pairs
Distance migrated