30 Questions
What is the purpose of the gene for the protein AraC in the DNA plasmid?
To regulate the expression of the GFP gene
What is the recognition site of the BsaI enzyme?
5'-GGTCTC-3'
What is the purpose of adding linkers to the prep work?
To replace the sticky ends created by BsaI
What is the ratio of oligos required for phosphorylation and annealing?
1:1
What is the purpose of adding DTT to the ligation reaction?
To maintain the reduced state of DNA ends
What is the purpose of heat shocking the bacteria?
To facilitate the uptake of the plasmid DNA
What is the purpose of the T4 PNK enzyme?
To phosphorylate the oligos
What is the purpose of the beta-lactamase (AmpR) gene?
To provide antibiotic resistance to the bacteria
What is the purpose of the T7 ligase enzyme?
To ligate the ds oligo into the vector
What is the purpose of plating the bacteria on selection agar plates?
To select for bacteria that have taken up the plasmid DNA
What is the primary function of the fluorescent protein components in the DNA plasmid?
To provide a visual indicator for successful transformation
What is the consequence of cutting the vector BPK764 with BsaI?
The formation of a sticky end
What is the purpose of phosphorylating the oligos in the preparation of the sgRNA?
To increase the efficiency of the ligation reaction
What is the outcome of the thermocycler protocol for the ligation reaction?
The ligation of the ds oligo into the vector
What is the role of the ATP in the ligation reaction?
To provide energy for the T7 ligase enzyme
What is the purpose of allowing the bacteria to sit with the oligos for 20 minutes before heat shocking?
To allow the uptake of the plasmid DNA
What is the consequence of not adding the linkers to the prep work?
The failure of the ligation reaction
What is the primary function of the vector BPK764?
To encode both the gRNA + Cas9
What is the outcome of the 6 cycles of the thermocycler protocol for the ligation reaction?
The ligation of the ds oligo into the vector
What is the purpose of using the T4 buffer in the phosphorylation and annealing reaction?
To provide a suitable environment for the T4 PNK enzyme
What is the likely outcome if the phosphorylation and annealing steps are not performed in a 1:1 ratio of oligos?
The ds oligo will not form properly
What is the purpose of adding molecular grade water to the sgRNA oligo stock solution?
To dilute the oligo to a working concentration
What would happen if the beta-lactamase (AmpR) gene was not present in the DNA plasmid?
The transformed bacteria would not survive on plates with ampicillin
What is the role of the 6 cycles of the thermocycler protocol for the ligation reaction?
To ligate the ds oligo into the vector
What would be the consequence of not adding the T4 PNK enzyme to the phosphorylation and annealing reaction?
The oligos would not be phosphorylated
What is the role of the ATP in the ligation reaction?
To provide energy for the ligation reaction
What is the purpose of the 95-degree step in the thermocycler protocol for the phosphorylation and annealing reaction?
To inactivate the T4 PNK enzyme
What would be the consequence of not using the T7 ligase enzyme in the ligation reaction?
The ligation reaction would not occur
What is the role of the BsaI enzyme in the preparation of the vector?
To cut the vector BPK764
What is the purpose of allowing the bacteria to sit with the oligos for 20 minutes before heat shocking?
To allow the uptake of the plasmid DNA
Study Notes
DNA Plasmids Components
- DNA plasmids contain cDNA for fluorescent proteins, including araC, which controls the GFP gene like an ON/OFF switch.
- GFP/mCherry are fluorescent proteins, and Beta-lactamase (AmpR) allows transformed bacteria to survive on plates with ampicillin.
Vector BPK764
- Vector BPK764 is used to encode both gRNA and Cas9.
- BsaI is used to cut out part of BPK764, with a recognition site of 5’-GGTCTC-3’, and it cuts 4 base pairs after on the opposite strands, forming a sticky end.
Preparing the Ordered Oligo
- Two oligo solutions were received, one for the forward strand and one for the reverse strand.
- An sgRNA oligo stock solution was created, with a concentration of 100uM, and 10uM was taken to add to 90uM of distilled water.
Phosphorylation and Annealing
- Phosphorylation of oligos and annealing of 2 strands to generate a double-strand DNA were done in the same step.
- Requirements include a 1:1 ratio of oligos, T4 buffer, T4 PNK, and molecular grade water.
- The reaction was performed in a thermocycler with the following conditions: 37°C for 30 minutes, 95°C for 5 minutes, and then cooled down to 25°C at 5°C/min.
Preparing the Vector and Ligation
- Cutting the vector and ligating the ds oligo into the vector were done in the same step.
- Two ligation reactions were set up, with pBK764 (pre-digested with BsaI), buffer, DTT, ATP, BsaI-HF, and T7 ligase.
- The reaction was performed in a thermocycler with 6 cycles of 37°C for 5 minutes, then 21°C for 5 minutes.
Bacterial Transformation
- The bacteria were allowed to sit with the oligos for 20 minutes, then heat shocked.
- The bacteria were then plated on selection agar plates.
Test your knowledge of DNA plasmids containing cDNA for fluorescent proteins, including components like araC, GFP/mCherry, and beta-lactamase. Learn about vector BPK764 and its role in encoding gRNA and Cas9.
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