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Gel Electrophoresis Lab. Analysis of Precut Lambda DNA. Restriction Enzymes. Our lambda virus DNA samples have been precut with three different restriction enzymes. L – lambda DNA P – Pst I lambda digest E – Eco RI lambda digest H – Hind III lambda digest. Running the Gel.
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Gel Electrophoresis Lab Analysis of Precut Lambda DNA
Restriction Enzymes Our lambda virus DNA samples have been precut with three different restriction enzymes. L – lambda DNA P – PstIlambda digest E – EcoRI lambda digest H – HindIII lambda digest
Running the Gel • The phosphate groups in the sugar-phosphate backbone of DNA are negatively-charged. • Molecules move through the gel at different rates, determined largely by their size and charge. Shorter molecules move through the gel faster than longer molecules.
Reasons for Each Step • Loading Dye Two sizes of blue molecules, small and large, help visualize movement of sample across gel. • Heat DNA Samples Denatures double stranded DNA to produce single strands that more easily move through gel.
Reasons for Each Step • Tap Tubes on Table Top moves sample to bottom of tube for collection • Run the Gel in Chamber electrical field pulls negatively charged DNA through the gel, with distance moved proportional to size • Stain Overnight stain attaches to DNA within gel, making bands of DNA visible
Lab Tips • Follow the “How to Use a Micropipet” instructions at your station. • When finished, eject the tip into the disposal at your station. • Carefully handle the DNA samples! • Be sure your gel is well-side up and fully submerged in the buffer solution. • Hold the micropipetjust over the well. Be careful not to puncture the bottom of the well with the micropipet! • Use tape to label which gel is yours (top or bottom).
