PCR
What are the steps of PCR and their function?
Denaturing- heat to separate DNA strands
Annealing- cool for primers to bind
Extension- heat for the polymerase to amplify the DNA region
(True/False): the pGLO plasmid contains ampicillin
FALSE; pGLO contains ampR (the ampicillin resistance gene)
The difference between a missense and a nonsense mutation?
Missense- codes for a diffrent AA
Nonsense- codes for a stop codon
The type of primers used in PCR?
DNA oligonucleotide primers
What was the purpose of using Chimera in this project?
Chimera was used to visualize the ACR5 and ACR3 proteins; taking a close look at the individual mutation sites and predicting the effect on the protein interactions based on the AA properties
How many PCR reactions do you need to prepare for your master mix if you have 9 samples?
11 reactions
(True/False): Transcription factors bind to the promoter region
TRUE
(True/False): mRNA is double-stranded
FALSE; mRNA is single-stranded
Is the below sequence a Terminal Inverted Repeat?
5' GACCAC-TE-GTCGTC 3'
NO
What are the differences and similarities between Taq polymerase and Q5?
Both are DNA polymerases
But Q5 has better proofreading than Taq (important for site-directed mutagenesis and sequencing)
What is the starting amount, in L, of water and 10x TBE required to make 5L of 1x TBE?
0.5L TBE and 4.5L water
Why are bacteria exposed to CaCl2 (salts) and heat-shocked in the transformation experiment?
To increase transformation efficiency (weaken the cell membrane to allow plasmid to be inserted)
Which polymorphism would have the most deleterious effect: SNP, 3bp deletion, or 1bp insertion?
A 1 base pair insertion because it would lead to a frameshift mutation
A mutation in gene B causes cancer, so you perform PCR on a patient with the mutation and a patient with the wild-type gene. You observe a 1kb band for both the wild-type and the mutated gene. What type of mutation is it and how can you identify it?
The mutation is most likely a SNP and can be identified by sequencing and then using BLAST to compare the mutant vs the wild-type.
What is the KLD reaction and what is it used for?
Kinase Ligase DpnI:
Kinase and Ligase: Phosphorylate and ligate the newly synthesized plasmid with the mutation.
DpnI: A restriction enzyme that specifically "cuts" the methylated parental plasmid at a specific site, ensuring only the mutated plasmid remains.
What is the difference between a positive and a negative control?
Positive control- used as a reference with a known result (expect to see a band)
Negative control- used to make sure there is no contamination (no bands expected)
(True/False): You conduct an experiment to transform E.Coli with a plasmid that contains the gDNA encoding insulin. After incubation, you can extract the cloned insulin protein to treat diabetes...
False; bacteria do NOT have a splicing mechanism (only cDNA)
The COVID-19 virus has an RNA genome. To test if someone is infected, what would you extract and do with the sample?
Extract RNA, use reverse transcriptase to make cDNA, then use PCR to test for infection with COVID
What was the goal of the ACR5 project?
To mutate specific Amino Acids in the ACR5 protein using site-directed mutagenesis and observe if they affect the protein's function or interaction with other proteins (such as ACR3)
What would be the effect of increasing the % density of an agarose gel for gel electrophoresis?
It would help separate the smaller DNA fragments better (vs decreasing the % density would help separate larger fragments more easily)
You transformed E.Coli with the plasmid pRED and plated on media with different antibiotics. You obtain the below results:
LB: Lawn
LB+ Kanamycin: 0 colonies
LB+ Tetracycline: 150 colonies
LB+ Hygromycin: 0 colonies
Which antibiotic resistance gene does the pRED plasmid contain and what would you expect if you plate LB+ KAN+ TET?
pRED has a tetracycline resistance gene; you would expect no growth (0 colonies) because the bacteria are NOT resistant to KAN.