Recombinant DNA Technology and Plasmid Cloning in Biotechnology
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81 Terms
What is recombinant DNA technology?
A set of techniques used to isolate specific DNA fragments and link them to different DNA regions or add them to a different host organism.
What is the purpose of recombinant DNA technology?
To mix and match specific DNA sequences to create DNA molecules not found in nature.
Why is recombinant DNA important?
It is a cornerstone of biotechnology and is used for basic research and practical applications such as medicine and agriculture.
What is an example of a medicine produced using recombinant DNA?
Insulin used by people with diabetes.
What is DNA cloning?
The process of isolating a gene or DNA sequence and producing many identical copies of it.
What is a plasmid?
A small, circular DNA molecule that is physically separate from the bacterial chromosome.
Where are plasmids commonly found?
Bacterial cells.
Are plasmids part of the bacterial chromosome?
No.
Can plasmids replicate independently?
Yes.
What genes do cloning plasmids often contain?
Genes for antibiotic resistance.
Why is antibiotic resistance useful on a plasmid?
It allows researchers to select bacteria that contain the desired plasmid.
What is selection in the context of plasmids?
The process of using an antibiotic or other mechanism to allow only cells carrying the desired plasmid to survive.
What is a cloning vector?
A plasmid used to make copies of a foreign DNA sequence.
What is another term for a cloning vector?
Vector.
What can be inserted into a plasmid?
A DNA fragment from any organism.
What is the origin of replication on a plasmid?
The site where DNA replication is initiated.
Why does a plasmid need an origin of replication?
So the plasmid can be copied inside the bacterial cell.
What is the selection mechanism on a plasmid?
Often a gene that provides resistance to an antibiotic.
What is a multiple cloning site?
A region of a plasmid where genes of interest can be easily inserted.
What are restriction sites?
Places where restriction enzymes can cut DNA and create double-stranded breaks.
What must a gene of interest have to be expressed?
The necessary regulatory genetic elements, including a correctly oriented promoter.
What is a promoter?
A regulatory DNA element that allows transcription to initiate.
What is recombinant plasmid DNA?
A plasmid containing an inserted foreign DNA sequence.
What happens when recombinant plasmid DNA is inserted into bacteria?
The plasmid can be replicated and passed to daughter cells.
Why grow bacteria containing recombinant plasmids in nutrient broth?
To produce large numbers of bacterial cells containing many copies of the recombinant plasmid.
What is E. coli used for in this lab?
E. coli is used as a host organism to store, maintain, and produce recombinant plasmids.
What is DH5svg?
The E. coli strain used in this lab for cloning and plasmid production.
What are pDonor (pD) and pDonorGuide (pDG)?
The two plasmids being purified for the CRISPR experiment.
Why are pD and pDG purified?
To provide plasmid DNA needed for the CRISPR experiment the following week.
What is a miniprep?
A procedure used to purify plasmid DNA from bacterial cells.
What is the main goal of this week's lab?
To purify plasmids from E. coli using a miniprep protocol.
How are plasmids harvested from bacteria?
The bacteria are broken open, DNA is isolated, and plasmids are separated from the main bacterial chromosomes and other cellular materials.
What is the purpose of growing E. coli cultures before a miniprep?
To produce enough bacterial cells containing the plasmid to purify.
What is LB liquid medium?
A nutrient-containing liquid medium used to grow bacteria.
Why does the LB medium contain spectinomycin?
To select for bacteria that contain the plasmid with spectinomycin resistance.
What is a 37°C shaking incubator used for?
To grow and prepare bacterial cultures under appropriate temperature and mixing conditions.
What is a tabletop centrifuge used for?
To separate materials based on density by spinning samples.
What is the purpose of biohazard waste?
To dispose of biological materials such as bacterial cultures safely.
What does Buffer P1 contain/do?
It helps resuspend the bacterial pellet; RNase A removes RNA.
What does RNase A do?
It breaks down RNA so that RNA does not contaminate the purified DNA.
What does Buffer P2 do?
It helps break open bacterial cells so plasmid DNA is released.
What is cell lysis?
The process of breaking open cells to release their contents.
What does detergent do during lysis?
It helps disrupt the bacterial cell membrane.
What does Buffer N3 do?
It helps neutralize the lysed sample and allows cellular debris and unwanted material to precipitate while plasmid DNA remains in solution.
What happens when the lysate is neutralized?
Cellular debris and unwanted materials precipitate, allowing plasmid DNA to remain in the liquid.
What is a spin column?
A tube containing a silica membrane that binds negatively charged plasmid DNA and allows other materials to be separated.
Why does DNA bind to the spin column?
The negatively charged DNA binds to the silica membrane under the appropriate conditions.
What does Buffer PE do?
It washes the DNA-bound column to remove contaminants and clean the DNA.
What is ethanol used for in the wash buffer?
It helps wash contaminants away while allowing DNA to remain bound to the column.
What does Buffer EB do?
It allows the negatively charged plasmid DNA to be released from the silica column for collection.
What is elution?
The process of releasing purified DNA from the spin column into a collection tube.
What is a NanoDrop?
A microvolume spectrophotometer used to measure nucleic acid concentration and purity.
How much sample does a NanoDrop require?
Only about 1-2 µL of sample.
How does a NanoDrop hold the sample?
A very small sample is placed directly on a pedestal and the upper arm is closed to create a sample column.
What wavelength do nucleic acids absorb most efficiently?
260 nm.
Why is 260 nm used to determine plasmid concentration?
Nucleic acids absorb light most efficiently at 260 nm.
What does the NanoDrop measure?
The absorbance of the sample to determine nucleic acid concentration and assess purity.
What is nucleic acid concentration?
The amount of nucleic acid present in a sample.
What is sample purity?
A measure of how free the DNA preparation is from contaminants.
Why are purity measurements important?
Contamination can cause overestimation of nucleic acid concentration and inaccuracies in downstream experiments or measurements.
What is the ideal 260/230 ratio for DNA?
Approximately 2.0.
What is the ideal 260/280 ratio for DNA?
Approximately 1.8.
What does a low 260/230 ratio indicate?
Contamination from protein or kit reagents may be present.
What does a low 260/280 ratio indicate?
Contamination from protein or kit reagents may be present.
What does the 260/280 ratio compare?
Absorbance at 260 nm to absorbance at 280 nm.
What does the 260/230 ratio compare?
Absorbance at 260 nm to absorbance at 230 nm.
What happens after measuring a sample on the NanoDrop?
The pedestal must be cleaned so it is ready for the next sample.
What is CRISPR?
A native bacterial defense system used to remove foreign invading DNA.
What does bacterial CRISPR naturally defend against?
Foreign DNA, such as DNA from invading phages.
What is CRISPR-Cas9?
A gene-editing tool that builds upon the natural bacterial CRISPR defense system.
What is the purpose of this week's plasmid purification in relation to CRISPR?
The purified plasmids will be used in the CRISPR-Cas9 gene-editing experiment the following week.
What is the relationship between plasmids and DNA cloning?
Plasmids can act as cloning vectors that carry and replicate DNA sequences of interest.
Why are bacteria useful for recombinant DNA technology?
They can replicate plasmids, producing many copies of recombinant DNA.
What happens to a recombinant plasmid when a bacterial cell divides?
The plasmid is replicated and passed to daughter cells.
Why is antibiotic selection important?
It ensures that cells growing in the culture carry the desired plasmid.
What are the major steps of a miniprep?
Grow plasmid-containing bacteria, harvest cells, lyse cells, neutralize, bind DNA to a spin column, wash the DNA, and elute purified plasmid DNA.
What is the overall goal of the miniprep buffers?
To break open cells, remove unwanted cellular materials, bind and wash the plasmid DNA, and finally release the purified DNA.
What is the difference between a bacterial chromosome and a plasmid?
The bacterial chromosome contains the main genetic information, while plasmids are smaller circular DNA molecules that are separate and can replicate independently.
What is the purpose of plasmid antibiotic resistance in cloning?
It allows researchers to select bacteria containing the desired plasmid from bacteria that do not contain it.
What are the two plasmids purified in this lab?
pDonor (pD) and pDonorGuide (pDG).
What will the purified plasmids be used for?
The CRISPR-Cas9 gene-editing experiment.