Questions and Answers
What pipette and what setting would you use to get 1000 microliters (1 mL)?
p1000 and 100
What pipette and what setting would you use to get 50 microliters (0.05 mL)?
p200 and 050
How to get the final percentage of each ingredient?
Take the ingredient total divided by the overall total.
What are the three kinds of agar plates used for in Week 5?
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What is the 50x TAE buffer used for in week 4?
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What is a plasmid?
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What are the three buffers in Week 3, and what are their functions?
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What charge is DNA and therefore what part of the box is it attracted to (cathode or anode)?
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What is the gel called in electrophoresis?
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What does the buffer do in gel electrophoresis?
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How to make 300 mL of 1X TAE buffer from 50X stock solution?
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What were the purposes of the three buffers we used in Week 3 during our DNA prep?
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How did we isolate DNA from the buffer/lysate mixture in Week 3?
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What role does the charge of DNA play in gel electrophoresis?
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How did we stimulate the E.coli cells to pick up the pGLO plasmid?
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What are the key components of the pGLO plasmid that allow for the formation of glowing colonies on the LB+Amp+Ara plates?
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What is genetic transformation?
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Name and describe the two types of ways to introduce DNA to a cell.
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What activates the pGLO plasmid at the promoter?
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What kind of growth was in the LB -pGLO plate?
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What kind of growth was in the LB/AMP -pGLO plate?
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What kind of growth was in the LB/AMP/ARA +pGLO plate?
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What kind of growth was in the LB/AMP +pGLO plate?
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Study Notes
Pipettes and Measurements
- Use a p1000 pipette set to 100 to measure 1000 microliters (1 mL).
- Use a p200 pipette set to 050 for measuring 50 microliters (0.05 mL).
Calculating Percentages
- Final percentage of each ingredient is calculated by dividing the ingredient total by the overall total.
Agar Plates in Week 5
- Three types of agar plates are used to promote bacterial growth.
50x TAE Buffer in Week 4
- 50x TAE buffer is utilized for preparing DNA gel.
Plasmids
- Plasmids are DNA pieces capable of existing independently from a cell's chromosome.
Buffers in Week 3
- MX1: Contains RNAse for degrading RNA.
- MX2: Contains detergent for disrupting bacterial cell membranes.
- MX3: Facilitates isolation of nucleic acids (DNA) from other cellular components.
DNA in Gel Electrophoresis
- DNA carries a negative charge, attracting it to the cathode during electrophoresis.
Gel in Electrophoresis
- The gel used in electrophoresis is agarose gel.
Function of Buffer in Gel Electrophoresis
- The buffer allows electric current to flow through the gel.
Preparing 1X TAE Buffer
- To make 300 mL of 1X TAE buffer from a 50X stock solution, dilute it accordingly.
Purposes of Buffers in DNA Preparation
- Buffers are used to degrade RNA, lyse cells, and precipitate large biomolecules out of solution.
DNA Isolation Method in Week 3
- DNA is isolated from the buffer/lysate mixture using a column that temporarily binds DNA.
Charge of DNA in Electrophoresis
- DNA's negative charge leads to its repulsion from the anode and attraction to the cathode.
E. coli Transformation Process
- E. coli cells are stimulated to take up the pGLO plasmid through temperature shifts that induce a fluid influx.
Components of pGLO Plasmid
- Key elements include the origin of replication, ampicillin resistance gene (Ampr), and arabinose promoter driving the GFP gene expression.
Genetic Transformation Definition
- Genetic transformation involves introducing foreign DNA into an organism or cell.
Methods of Introducing DNA to Cells
- Electroporation: Applying an electric current to the sample.
- Chemical transformation: Heat shock treatment of cells in the presence of calcium.
Activation of pGLO Plasmid
- Arabinose activates the pGLO plasmid at its promoter, enhancing transcription of the GFP gene.
LB -pGLO Plate Growth
- Displays a bacterial lawn without glowing colonies.
LB/AMP -pGLO Plate Growth
- No growth is observed; ampicillin eliminates bacteria that lack pGLO.
LB/AMP/ARA +pGLO Plate Growth
- Glowing individual colonies are present due to successful transformation.
LB/AMP +pGLO Plate Growth
- Displays individual colonies, but they do not glow indicating absence of arabinose for GFP expression.
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