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BIOL Exam I - Ex. 6
Gel Electrophoresis and Bacterial Transformation Analysis
| Question | Answer |
|---|---|
| How did we examine the size of our D1S80 amplicons? | by using agarose gel electrophoresis |
| How did we examine the size of the pGLO restriction fragments? | by using agarose gel electrophoresis |
| amplicon | - a piece of DNA or RNA that is the source and/or product of natural or artificial amplification or replication events - we made amplicons via PCR |
| Why were two different gels made (one 0.8% and the other 2.0%)? | - smaller DNA segments, like DNA amplicons, need thicker gel so that they do not run off into the buffer solution - larger DNA segments, like pGLO restriction fragments, need less dense gel so that they can move easier |
| During gel electrophoresis, you want the DNA molecules to run about 2/3 of the way through the gel before you turn it off. How can you tell when it gets to that point? | - loading dye and glycerol are mixed with the DNA samples - the dye helps you track the dye by making them a certain color, not transparent - the glycerol makes the DNA molecules sink into the gel better so they dont run off into the buffer |
| The pGLO plasmid has one ___ restriction site and one ___ restriction site. So, after the double digest you should see ___ fragment(s). Undigested plasmids can be supercoiled or open circles. | - EcoRI - BstEII-HF - two fragments |
| Only the ___ form of plasmid DNA runs according to its size (in kbp). | linear |
| What is an LED Transilluminator? What needs to be mixed with agarose gel in order to see bands? | - a device that projects LED light through a translucent sample for observation - Syber-Safe DNA stain (makes the bands glow) |
| Describe the resulting gel (restriction digest). What were in the wells? What did the bands look like? | - 1st well: the ladder - 2nd: pGLONE, open floppy circle or supercoil - 3rd: pGLORE, 2 fragments -4th: positive control digest, 1 linear band |
| Describe the resulting gel (PCR). What were in the wells? What did the bands look like? | - 1st well: the ladder - 2nd: negative control, no bands present - 3-6: students DNA - 7th: positive control, known template, 1 band |
| Looking at the result of gel electrophoresis (using PCR products), how would you determine genotype? How would you determine the alleles' actual sizes? | - two bands in a lane = heterozygous, one band in a lane = homozygous - use ladder to determine base pairs (length) - use base pairs to determine allele (using the chart) |
| So, what were the three main things you did in exercise 6? | - agarose gel electrophoresis of PCR products (from cheek cells) - agarose gel electrophoresis of products from the restriction digest of the pGLO plasmid - examine the agar plates that had bacteria (some transformed with pGLO) |
| transformation efficiency | - the number of colonies that were successfully transformed with a specific plasmid (like pGLO) - usually very low |
| To melt solidified agarose in a bottle in the microwave, heat it ___. | in short intervals of 15-20 seconds each |
| When microwaving a congealed solution of agarose, make sure to ___. | keep the cap on the bottle open |
| Removing the comb from a gel is made much easier, with less possibility of tearing the gel, if ___. | the area around the teeth of the comb is flooded with water |
| Upon its removal from the casting frame, a gel tore apart at the wells. What most likely explains how this gel was improperly cast causing it to break at the wells and became unusable? | the comb was inserted incorrectly at the adjustable end |
| DNA, because it has a _____ charge, moves to the _____ end (e.g., anode) of the field in gel electrophoresis. _____ DNA molecules migrate the most quickly. | - negative - positive - smaller |
| What is the quickest way to know if agarose gel is correctly oriented in an electrophoresis rig? | the wells can RUN TO RED (so they are closest to the black cathode) |
Created by:
jessica.gvc
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