5-week project
What was the end goal of our 5-week project?
To molecularly clone our cited1 genes, wild type (control) and clone 4 (cited1 knockout)
If you have 25 mg/mL of BSA and you need a total volume of 330 mL, how much BSA will you need? (BSA – formula weight is too big to list, comes as a solid)
8.25 g or 8250 mg BSA
What does western blotting detect?
Proteins
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
P200
1
5
6
_____ ul
156 ul
Transformation
If you have 15 mg/mL of BSA and you need a total volume of 1.5 L, how much BSA will you need? (BSA – formula weight is too big to list, comes as a solid)
22.5 g or 22500 mg BSA
If the primary antibody is derived from rabbit, what must the secondary be derived from?
rabbit, so anti-rabbit
Where did the concept of CRISPR-Cas9 gene editing originate from?
Bacterial immune response (discovered first in E.coli)
What is the organism from which phalloidin is obtained?
Death Cap mushroom
Following our 5-week cloning project, what was the flow of experiments to get to the final product
PCR > Gel Extraction & Purification > Ligation > Transformation > Miniprep > Restriction Digest > Gel Electrophoresis for confirmation
Assume you have a primer stock that is 30 µM. How much of your stock will you need to get a 1 µM concentration of primer into a 50 µL PCR reaction volume?
1.67 ul primer stock
Why is beta actin called a housekeeping gene?
Because of its broad and consistent expression across all eukaryotic cell types, and because expression levels of this protein is not affected by most experimental treatments
Explain Homology-Directed Repair
Precise DNA repair pathway that uses a homologousDNA template to fix double-strand breaks
F-Actin vs G-Actin?
G-Actin - actin subunits
F-Actin - filamentous actin (polymerized G-Actin)
How are restriction sites added to genetic sequences and plasmids?
Through the incorporation of restriction enzyme-specific sequences within the designed primers during PCR
If you have 1.8% Triton and you need a total volume of 500 mL, how much Triton do you need? (Triton – 625 g/mol, comes as a liquid)
9 mL Triton
How does IFN-gamma affect STAT1?
STAT1 is activated by IFN- 𝛾 to regulate immune responses. When IFN-𝛾 binds to its receptor, it triggers a cascade involving Janus kinases(JAKs), which in turn phosphorylate STAT1, allowing it to move to the nucleus and control gene expression to fight infections.
Explain Non-Homologous End Joining
DNA repair pathway that directly rejoins the ends of a broken DNA double-strand break without using a homologous template
What is meant by the term "miniprep"?
It is a small-scale preparation of DNA from bacterial cells
Explain the concept of blue-white screening
DNA insert is ligated in between the LacZ gene within our particular plasmid.
LacZ encodes for an enzyme called β-galactosidase
β-galactosidase cleaves X-gal, an analog of lactose
When X-gal is cleaved, it yields a blue precipitate that can be seen by the naked eye
If you need 330 mL of a solution, and you have 50mM of NaCl, how much NaCL would you need? (NaCl - 58.4 g/mol, comes as a solid)
0.96 g NaCl
List and describe the function for each component in Laemmli loading buffer
SDS - coats the proteins in a negative charge, denatures
B-ME - breaks disulfide bonds
Glycerol - increases sample density
Bromophenol blue dye - loading dye that tracks the sample through the gel
Describe in full the bacterial immune response that the CRISPR-Cas9 gene editing system was derived from
1. Foreign phage DNA inserted into a bacterial cell can be captured and integrated into its own genetic sequence called CRISPR arrays
2. If the bacteria encounters the viral DNA again, the bacteria will produce RNA segments from those CRISPR arrays they stored previously. These RNA segments (guide RNAs) will recognize and bind to specific regions of that intruding viral DNA
3. The bacteria then use cas9(CRISPR associated protein 9)or a similar enzyme to cut the viral DNA, disabling the virus
What type of bond is formed between complementary base pairs in DNA?
Hydrogen bonds