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Gel Ele
| Question | Answer |
|---|---|
| What is the purpose of ethidium bromide (EtBr) in gel electrophoresis? | It intercalates into DNA and fluoresces under UV light, allowing visualization of DNA bands |
| What percentage agarose gel is appropriate for the DNA fragments in this restriction digest? | 1% agarose gel |
| What is the purpose of gel loading buffer (GLB)? | Contains tracking dye and increases sample density so it sinks into the wells; also contains glycerol or sucrose (40%) and bromophenol blue dye (0.25%) |
| What temperature and duration are used for the restriction enzyme digestion? | 37°C for 40-45 minutes |
| Why must you "pulse spin" tubes after adding reagents? | To bring all liquid to the bottom of the tube, ensuring proper mixing |
| What factors determine the rate of DNA migration through an agarose gel? | Size and shape of the molecule; smaller molecules and compact molecules move faster than large, loose ones |
| What is the recognition sequence for HindIII? | 5' AAGCTT 3' / 3' TTCGAA 5' (produced by Haemophilus influenzae) |
| What is the recognition sequence for EcoRI? | 5' GAATTC 3' / 3' CTTAAG 5' (produced by Escherichia coli) |
| What happens to circular plasmid DNA when cut by ONE restriction enzyme? | It becomes linearized (one linear piece) |
| What happens to circular plasmid DNA when cut by TWO restriction enzymes? | It produces two linear fragments |
| Why does DNA migrate toward the anode (positive terminal) in gel electrophoresis? | Because DNA is negatively charged (it's an anion) and moves toward the positive terminal |
| How do closed circular plasmids compare to open circular (nicked) plasmids in migration speed? | Closed circular (supercoiled) plasmids move FASTER than open circular plasmids of the same size |
| How much sample is loaded into each well? | Approximately 15 μl of sample (after mixing with 2 μl GLB from the 20 μl total) |
| What two plasmids are being compared in this experiment? | pUC119 (3.2 kb) and pRU4x92 |
| Where are the EcoRI and HindIII restriction sites located on both plasmids? | In the multiple cloning site (MCS) of each plasmid |
| How can you identify which plasmid you isolated based on the gel results? | By comparing the number and sizes of fragments from each digest to the predicted pattern; each plasmid will have different fragment sizes when cut with the same enzymes |
Created by:
smurtab