IS3300 Electrophoresis and DNA Fragment Length Determination
Restriction enzymes are enzymes that are capable to cleaving, or cutting, DNA molecules in very specific spots based on the DNA molecule’s sequence of bases. Their discovery and isolation in the latter half of the 20th century provided scientists with an invaluable tool and rapidly expanded in the field of molecular biology. Agarose electrophoresis is a technique for separating the pieces of DNA that result from the use of restriction enzymes. In this activity students will perform agarose electrophoresis on three DNA samples that have already been treated with restriction enzymes. One sample has been cut with the enzyme HindIII, one has been cut with the enzyme EcoRI, and one has been cut with both enzymes. The resulting pattern of DNA bands on the agarose gel will show that each enzyme affects the DNA differently. Students will not only learn about the process of DNA electrophoresis but learn the techniques associated with the process, such as setting up an agarose gel, loading DNA samples in the agarose gel, and staining the gel in order to visualize the DNA bands. They will also learn how to determine the sizes of unknown DNA fragments after examining their results.
The kit includes specially-treated DNA samples that do not require refrigeration or freezing, prepared agarose that may simply be melted in a hot water bath or microwave, TBE electrophoresis buffer, and DNA stain. There is enough DNA to run 10 gels.
200mL Prepared Agarose, 0.8% Solution
500mL Tris Borate ETA Buffer, 5X
60mL DNA Stain, 20X
100 µL Lambda DNA/EcoRI Digest with Loading Dye
100 µL Lambda DNA/HindIII Digest with Loading Dye
100 µL Lambda DNA/EcoRI,HindIII Digest with Loading Dye
Needed but not supplied are agarose electrophoresis chambers, power supplies, and micropipettes capable of measuring 10µL.