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Questions and Answers
What is the purpose of using ampicillin in the replica plating process?
What is the purpose of using ampicillin in the replica plating process?
In the context of the Lac Z gene, what color will colonies appear if they contain a recombinant plasmid?
In the context of the Lac Z gene, what color will colonies appear if they contain a recombinant plasmid?
What type of tool is mentioned for transferring colonies in the screening process?
What type of tool is mentioned for transferring colonies in the screening process?
What is the role of beta-galactosidase in the screening process?
What is the role of beta-galactosidase in the screening process?
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What characteristic of the colonies indicates that they successfully incorporated foreign DNA?
What characteristic of the colonies indicates that they successfully incorporated foreign DNA?
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What is the purpose of treating E. coli cells with CaCl2 in the transformation process?
What is the purpose of treating E. coli cells with CaCl2 in the transformation process?
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Which of the following methods can be used for introducing DNA into a host cell?
Which of the following methods can be used for introducing DNA into a host cell?
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What role does the ligation solution play in the recombinant transformation process?
What role does the ligation solution play in the recombinant transformation process?
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What is the purpose of incubating competent cells on ice during transformation?
What is the purpose of incubating competent cells on ice during transformation?
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Electroporation is used to:
Electroporation is used to:
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Why is it necessary to place the tube back on ice immediately after heat shocking the competent cells?
Why is it necessary to place the tube back on ice immediately after heat shocking the competent cells?
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What is one common type of selection marker used in gene transfer?
What is one common type of selection marker used in gene transfer?
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What is the final step after adding the nutrient broth to the competent cell tube?
What is the final step after adding the nutrient broth to the competent cell tube?
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What is the primary purpose of incubating the cell mixture in a water bath for 3-4 hours?
What is the primary purpose of incubating the cell mixture in a water bath for 3-4 hours?
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Why is it important to include antibiotics in the LB agar plates?
Why is it important to include antibiotics in the LB agar plates?
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What is the significance of the pelleting and resuspending steps in the protocol?
What is the significance of the pelleting and resuspending steps in the protocol?
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What characteristics do the bacteria with successful transformation exhibit on antibiotic plates?
What characteristics do the bacteria with successful transformation exhibit on antibiotic plates?
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What role do selectable markers play in the transformation process?
What role do selectable markers play in the transformation process?
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What is the purpose of including a second antibiotic-resistant gene when using tetracycline and ampicillin?
What is the purpose of including a second antibiotic-resistant gene when using tetracycline and ampicillin?
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Why is it essential to ensure sterility when spreading the cell suspension on the agar plate?
Why is it essential to ensure sterility when spreading the cell suspension on the agar plate?
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What will occur if non-transformed cells are plated on agar with the relevant antibiotic?
What will occur if non-transformed cells are plated on agar with the relevant antibiotic?
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Study Notes
Lecture 4 - Methods of Gene Transfer
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Lecture Contents:
- Methods of recombinant gene delivery
- Bacterial transformation
- Electroporation
- Types of selection markers
- Antibiotic resistance genes
- LacZ gene
Learning Outcomes
- Students will be able to describe the methods of gene transfer into host cells.
- Students will be able to describe different selection markers used to identify positive clones.
Bacterial Transformation
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Methods of introducing free DNA:
- CaCl₂-heat transformation: Bacteria cells treated with ice-cold CaCl₂ and then exposed to high temperature (42-45°C) for 45-90 seconds
- Electroporation: Using an electric field to render the host cell wall permeable to free DNA.
CaCl₂ or Heat Transformation Concept
- E. coli bacterial cells are treated with CaCl₂ to allow plasmid attachment.
- Plasmids are co-incubated with bacteria in a microtube for DNA transfer.
Recombinant Transformation (Procedure)
- 1. Prepare competent cells: Competent cells are stored frozen at -80°C.
- 2. Thaw and resuspend: When needed, thaw competent cells on ice and flick to resuspend.
- 3. Add ligation solution: Open the competent cell tube and add 4 µL of ligation solution to the tube.
- 4. Close tube, keep on ice: For 30 minutes, place the tube on ice.
- 5. 42°C bath (45 seconds): Remove the tube from ice and immediately hold it in a 42°C water bath for 45 seconds.
- 6. Return to ice (1 minute): Place the tube directly back on ice for 1 minute.
- 7. Add LB broth: Use a sterile pipette to add 10 drops of sterile LB nutrient broth to the competent cell tube.
- 8. Mix and invert: Gently mix by tipping the tube and inverting it.
- 9. Incubate at 37°C: Incubate the mixture for 3-4 hours in a water bath or on a 37°C shaker for 45 minutes.
- 10. Plate the cells: Label LB agar plates with name and date, and add appropriate antibiotics.
- 11. Spread cells on agar: Pellet cells, resuspend with supernatant, place cell suspension in the center of each agar plate, and spread evenly using a sterile spreader.
- 12. Incubate the plates: Incubate the plates (agar down) for 2 hours, then invert (agar up) and incubate for 24-36 hours in a 37°C incubator.
Why Use Selectable Markers?
- Required due to ligation and bacterial transformation inefficiencies (even with high-efficiency systems, only a small fraction of bacterial cells take up the DNA).
- Prevent non-transformed cells from growing.
- Differentiate transformed cells containing plasmids with DNA inserts from those without (using additional tests).
Antibiotic Resistance Genes
- Used to identify successful transformation.
- Transformants are the only cells growing on an agar plate with the relevant antibiotic.
- A second antibiotic-resistant gene can be used to check for foreign DNA insertion.
- Example: Tetracycline and ampicillin resistance plasmids: first grow cells with tetracycline, then switch to ampicillin as only those with the correct insert survive.
- Use replica plating to confirm foreign DNA insertion.
Selectable Markers: LacZ Gene
- Use agar containing an antibiotic and X-gal.
- The LacZ gene produces beta-galactosidase.
- Beta-galactosidase converts X-gal to a blue product (Indole blue).
- Bacteria with the plasmid and intact LacZ gene form blue colonies.
- Bacteria with a disrupted LacZ gene (and an insert) form white colonies (no blue product).
- White colonies indicate recombinant plasmids.
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Description
This quiz covers the various methods used for gene transfer, including bacterial transformation and electroporation. Students will explore selection markers such as antibiotic resistance genes and LacZ. By understanding these methods, learners will gain insights into recombinant gene delivery processes.
