20 Questions
What is the purpose of adding additional bases at the 5' ends of both oligos?
To ensure the DNA oligos can be cloned into a vector
What is the advantage of designing a complementary strand and flipping it to make the reverse strand?
It increases the likelihood of Cas9 DNA cleavage
What is the format of the cDNA sequence used to design the gRNA?
FASTA format
What is the purpose of using IDT to get custom gRNA?
To get a selection of target sequences with on-target scores
What is the purpose of using a bacterial expression plasmid containing a cDNA for fluorescent protein?
To allow for fluorescence when transformed into bacteria
What is the primary function of the Cas9 protein in the described experiment?
To cut out specific DNA sequences
What is the purpose of using a complementary strand and flipping it to make the reverse strand?
To ensure efficient targeting of both strands of DNA
What type of sequence is used to design the gRNA?
cDNA sequence
What is the purpose of adding the 'atag' sequence to the forward oligo?
To facilitate cloning into a vector
What is the purpose of using IDT to get custom gRNA?
To obtain a selection of target sequences with on-target scores
What is the expected outcome when a bacterial expression plasmid containing a cDNA for fluorescent protein is transformed into bacteria?
The bacteria will fluoresce
What is the significance of the on-target score provided by IDT for the target sequences?
It predicts the efficacy of the gRNA in gene knockout
What is the purpose of designing a DNA oligo for the Cas9 protein?
To create a custom gRNA for gene knockout
What is the advantage of using a cDNA sequence for fluorescent protein in the design of the gRNA?
It provides a template for the design of the gRNA
What is the expected outcome of using the Cas9 protein to cut out the fluorescent protein gene?
The fluorescent protein gene will be knocked out
What is the primary purpose of using a fluorescent protein in the experiment?
To identify potential targets for the gRNA
What is the significance of using both forward and reverse strands in the design of the DNA oligo?
It ensures efficient targeting of the gene by the Cas9 protein
What is the role of the on-target score provided by IDT?
It selects the optimal target sequence for the gRNA
What is the purpose of transforming the bacterial expression plasmid into bacteria?
To express the fluorescent protein in the bacteria
What is the ultimate goal of designing gRNAs to knockout fluorescent protein genes?
To create a bacterial strain that does not express the fluorescent protein
Study Notes
Bacterial Expression Plasmids
- Contain cDNAs for fluorescent proteins (EGFP or mCherry), leading to fluorescence when transformed into bacteria
gRNA Design
- Used IDT to design custom gRNA for chosen protein
- Imported cDNA sequence in FASTA format
- Received a selection of target sequences from IDT, each with an on-target score
Oligo Design for Cas9 Protein
- Used target sequence as forward strand
- Designed complementary strand and flipped it to make the reverse strand for efficient targeting
- Added additional bases at the 5’ ends of both oligos for cloning into a vector
- Forward oligo: added atag
- Reverse oligo: added aaac
Practical Overview
- Designed gRNAs to knock out fluorescent protein genes using Cas9 protein
This quiz covers the design of bacterial expression plasmids, gRNA design, and oligo design for Cas9 protein. Learn about CRISPR-Cas9 gene editing and its applications in molecular biology.
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