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Questions and Answers
The boiling method is a classic alternative to the alkaline lysis method for plasmid DNA preparation.
The boiling method is a classic alternative to the alkaline lysis method for plasmid DNA preparation.
True
Lysozyme can be stored at room temperature for plasmid DNA preparation.
Lysozyme can be stored at room temperature for plasmid DNA preparation.
False
The boiling step in the procedure lasts for exactly 30 seconds.
The boiling step in the procedure lasts for exactly 30 seconds.
False
STET is composed of 50 mM Tris-Hcl, pH 8.0, and 8% sucrose.
STET is composed of 50 mM Tris-Hcl, pH 8.0, and 8% sucrose.
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Isopropanol is added to the bacterial pellet after centrifugation.
Isopropanol is added to the bacterial pellet after centrifugation.
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Plasmids are double-stranded, linear DNA found in bacteria.
Plasmids are double-stranded, linear DNA found in bacteria.
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Plasmids can be transferred between bacteria of the same species only.
Plasmids can be transferred between bacteria of the same species only.
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The size of plasmids can range from 1 to 1000 kilo base pairs.
The size of plasmids can range from 1 to 1000 kilo base pairs.
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Cell lysis during plasmid extraction is facilitated by sodium hydroxide and SDS.
Cell lysis during plasmid extraction is facilitated by sodium hydroxide and SDS.
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Potassium acetate is used in the neutralization step to increase alkalinity.
Potassium acetate is used in the neutralization step to increase alkalinity.
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Glucose is included in the re-suspension solution to prevent cell bursting.
Glucose is included in the re-suspension solution to prevent cell bursting.
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Cell debris remains mixed with plasmid DNA after centrifugation.
Cell debris remains mixed with plasmid DNA after centrifugation.
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Vigorous mixing is encouraged during the lysis phase to shear genomic DNA.
Vigorous mixing is encouraged during the lysis phase to shear genomic DNA.
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The procedure starts with the inoculation of rich medium with a single colony of transformed bacteria.
The procedure starts with the inoculation of rich medium with a single colony of transformed bacteria.
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Ethanol is used to wash the pellet after nucleic acid precipitation.
Ethanol is used to wash the pellet after nucleic acid precipitation.
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Alkaline lysis solution I is added after the neutralization step.
Alkaline lysis solution I is added after the neutralization step.
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Centrifugation is performed at maximum speed for 30 minutes at 4°C in the initial step.
Centrifugation is performed at maximum speed for 30 minutes at 4°C in the initial step.
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Phenol-chloroform extraction is an optional step in the procedure.
Phenol-chloroform extraction is an optional step in the procedure.
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The DNA is stored at a temperature of -40°C.
The DNA is stored at a temperature of -40°C.
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150 μl of ice-cold Alkaline lysis solution III is added after the addition of Alkaline lysis solution II.
150 μl of ice-cold Alkaline lysis solution III is added after the addition of Alkaline lysis solution II.
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Ethanol evaporation takes approximately 15-20 minutes.
Ethanol evaporation takes approximately 15-20 minutes.
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Study Notes
Plasmid DNA Isolation
- Plasmids are double-stranded, circular, extrachromosomal DNA found in bacteria
- They are used in recombinant DNA experiments
- Plasmids are used for cloning genes from other organisms
- Plasmids enable large-scale production of foreign DNA
- Plasmids can be transferred between bacteria, within the same species or different species
- Plasmids range in size from 1 to 1000 kilobase pairs
- Plasmids are considerably smaller than the chromosomal DNA within a bacterium
- Bacterial chromosomal DNA contains several million base pairs
Alkaline Lysis Method for Plasmid DNA Extraction
- Cell Growth and Harvesting: Bacterial culture is grown to sufficient density
- Cells are pelleted via centrifugation to separate from the growth medium
- Re-suspension: Cell pellet is re-suspended in a solution (Solution I) containing Tris, EDTA, glucose, and RNase
- Divalent cations (Mg2+, Ca2+) are crucial for DNase activity and cell wall integrity
- EDTA chelates divalent cations to prevent DNase damage and destabilizes the cell wall
- Glucose maintains osmotic pressure to prevent cell bursting
- RNase degrades cellular RNA
- Lysis: Lysis buffer (Solution II) contains sodium hydroxide (NaOH) and the detergent SDS
- SDS disrupts the cell membrane
- NaOH breaks down the cell wall and denatures DNA
- Double-stranded DNA (dsDNA) is converted into single-stranded DNA (ssDNA)
- Avoid vigorous mixing to prevent shearing of genomic DNA
Neutralization
- Addition of potassium acetate (solution III) reduces alkalinity
- Hydrogen bonding between bases of ssDNA is re-established, allowing selective renaturation of plasmid DNA
- Avoid vigorous mixing to prevent re-annealing of sheared genomic DNA
Separation and Cleaning
- Precipitation of denatured cellular proteins, SDS, and single-stranded genomic DNA occurs
- Centrifugation separates the white precipitate from plasmid DNA
Cleaning and Concentration
- Plasmid DNA remains in a solution with salt, EDTA, RNase, and residual cellular proteins
- Cleaning and concentration methods like phenol/chloroform extraction followed by ethanol precipitation are used
Procedure
- Inoculation: Inoculate 2 ml of rich medium/broth with antibiotic using a single transformed bacterial colony
- Incubate overnight at 37°C with vigorous shaking
- Centrifugation: Pour 1.5 ml of overnight culture into a microfuge tube and centrifuge at maximum speed for 30 seconds at 4°C
- Medium Removal: Aspirate the medium leaving the bacterial pellet dry
- Resuspension: Vigorously vortex the pellet in 100 µl of ice-cold alkaline lysis solution I
- Alkaline Lysis: Add 200 µl of freshly prepared Alkaline lysis solution II to each bacterial suspension and tightly close the tube and invert to mix well; store in ice
Neutralization
- Add 150 µl of ice-cold alkaline lysis solution III , close the tube, and gently invert several times
- Store in ice for 3-5 minutes
Centrifugation
- Centrifuge the bacterial lysate for 5 minutes at maximum speed at 4°C
- Collect the supernatant into a fresh tube
(Optional) Phenol-Chloroform Extraction
- Optionally add an equal volume of phenol:chloroform
- Mix organic and aqueous phases by vortexing then centrifuge at maximum speed for 2 minutes at 4°C
- Transfer supernatant into a fresh tube
Nucleic Acid Precipitation
- Add 2 volumes of ethanol at room temperature to precipitate nucleic acids
- Vortex the solution, then let it stand 2 minutes at room temperature
- Centrifuge at maximum speed for 5 minutes at 4°C
Supernatant Removal
- Aspirate the supernatant and invert the tube to drain remaining liquid
Ethanol Wash
- Add 1 ml of 70% ethanol to the pellet, invert the closed tube, and recover DNA by centrifugation
Supernatant Removal
- Aspirate the supernatant, taking care the pellet may not adhere tightly to the tube
Ethanol Evaporation
- Remove any ethanol beads and allow the open tube to stand at room temperature until ethanol evaporates (5-10 minutes)
Storage
- Dissolve nucleic acids in 50 µl of TE (pH 8.0) containing 20 µg/ml DNase-free RNase A
- Gently vortex the solution and store DNA at -20°C
Boiling Method for Plasmid DNA Preparation
- The boiling method is an alternative to alkaline lysis for plasmid DNA preparation
- It follows the same principles as alkaline lysis
- Materials: STET, Lysozyme, Isopropanol, 70% ethanol, TE, Boiling water bath.
- (Specific concentrations and ratios of components in the solutions are not provided in the summarized text).
Methods for Plasmid DNA Extraction Using Boiling Method
- Inoculation and Culture Setup: Inoculate 2-3 ml broth containing antibiotic with bacterial colony; incubate at 37°C overnight
- Bacterial Cell Harvest: Harvest bacterial cells by centrifugation for 2 minutes at 10,000 rpm; remove supernatant and leave pellet
- STET Addition and Resuspension: Add 200 µl of STET to each tube with the pellet; Resuspend cells by vortexing
- Boiling Step: Immediately place tubes in boiling water for exactly 45 seconds
Centrifugation and Pellet Formation
- Centrifuge tubes at 10,000 rpm for 10 minutes to form a large, sticky pellet
Pellet Removal and Isopropanol Addition
- Remove pellet using a wooden toothpick
- Add 200 µL of isopropanol to each tube; centrifuge at 10,000 rpm for 5 minutes
Supernatant Aspiration and Ethanol Wash
- Aspirate supernatant
- Wash the pellet with 500 µL of 70% ethanol
- Centrifuge for 1 minute to compact the pellet
Pellet Drying and Resuspension
- Air-dry the pellet for 10 minutes
- Resuspend each pellet in 100 µL of TE buffer
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Description
This quiz covers the fundamentals of plasmid DNA isolation, including their role in recombinant DNA experiments, gene cloning, and large-scale production of foreign DNA. It also details the alkaline lysis method for plasmid DNA extraction, focusing on cell growth, harvesting, and the re-suspension process. Test your knowledge on these critical biotechnology techniques!
