Microbiology and Molecular Biology Techniques

Flashcards generated from lecture notes to aid in exam preparation, focusing on key concepts and definitions related to microbiology and molecular biology techniques.

What is the biological significance of Salmonella in the Ames test?

Mutant Salmonella bacteria cannot grow and reproduce their own histidine, making them useful for detecting mutagens.

What is the importance of 'his' genes in the Ames test?

'his' to 'hist-' mutants revert to allow detection of potential mutagens.

Briefly describe how the Ames Test works.

Expose a bacterial strain to a test compound, plate on histidine-free medium, and observe growth to identify potential mutagens.

Why is aseptic technique important when working with bacteria or solutions?

To avoid cross-contamination.

What is the purpose of EDTA in DNA purification?

Removes Mg2+ and Ca2+, protects DNA from DNases, and destabilizes the cell wall and outer membrane.

What is the role of SDS in purifying DNA?

It is a detergent that solubilizes lipids in the outer and inner membranes.

What is the purpose of Phenol in the process of purifying DNA?

Denaturing agent that removes proteins from the cell lysate.

What role does Chloroform play in purifying DNA?

Solubilizes lipids and promotes clear phase separation.

Why is Ethanol used in DNA purification?

To cause DNA to precipitate out of solution.

What is the function of Proteinase K in DNA purification?

Removal of protein and other cell debris.

What is the purpose of NaCl in DNA purification?

Prevents protein from binding to DNA.

What is the function of RNAse A?

Enzyme that removes RNA from DNA.

What is the purpose of Tris Buffer in DNA purification?

Keeps pH stable.

Why add Sodium Acetate to DNA precipitation?

To clump DNA and neutralize the negative charge of DNA.

What is the purpose of agar in bacteriological media?

Separates DNA from other cellular components.

What is the purpose of top agar with histidine in reversion frequency experiments?

Allows cells time to absorb potential mutagens.

What is the significance of absorbance at 260 nm when using a spectrophotometer?

The wavelength at which nucleic acids absorb the most light.

What does a 260/280 ratio of 1.7-2.0 indicate?

The DNA sample is pure.

What enzyme is used in Polymerase Chain Reaction (PCR)?

Taq Polymerase

What occurs during the 94-96°C step in PCR?

Separation, denaturing of DNA.

What is the annealing temperature range in PCR?

50-55°C

What occurs during the 60-72°C step in PCR?

Extension and elongation.

What is the function of dideoxynucleotides?

Modified nucleotide that stops DNA chain elongation.

Where is the mutation located if it is HisG - TA1535?

Base substitution.

Where is the mutation located if it is HisB - TA1538

Frameshift-deletion of C.

Why are VBM plates selective?

Only his+ cells can form colonies.

What are the criteria for being Mutagenic?

If 2x reversion frequency of negative control

Why is salt (sodium acetate) added to DNA precipitation?

To clump DNA together because they usually repel each other.

What is the function of ethanol in DNA precipitation?

Causes DNA to precipitate out of solution.

What is the PAM site sequence?

5'-NGG-3'

What is the composition of Guide RNA in CRISPR-Cas9?

5'-N20-3' (20 base pairs, 40-80% GC content), not including PAM site.