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BIOL Exam I - Ex. 6

Gel Electrophoresis and Bacterial Transformation Analysis

QuestionAnswer
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