Lab Quiz 2 Flashcards
What are autotrophs
Bacteria have mutations that prevent them from synthesizing compounds needed for cell growth
What is a reverse mutation?
Can restore the ability of the bacterium to synthesize the essential compound
What is a prototroph
A bacteria which can grow without any nutrient supplementation
What are spontaneous mutations
Mutation events occurring in the absence of a mutagen. Can cause a lost gene to reappear
What is a negative control
A plate without the use of a mutagen. The average number of colonies on these control plates measure the rate of spontaneous mutation
What are positive control plates
Include the mutagen which are known to produce revertant colonies in the mutant bacterial strain
What is methyl methane sulfonate
Used for positive control, known as a mutagen
What was DMSO used for
Negative control, was used as a solvent for student sample. Needs to be negative control in order to ensure this isnt causing the mutations
What is the sterility control
Asses the sterility of each student substance
How do we know if a substance is toxic?
It requires a zone of inhibition
How do we know if a substance is mutagenic?
Has to be double number of colonies from negative control
What are the two types of mutagens?
Direct acting mutagens react directly with DNA to produce base changes
Indirect-acting mutagens are not themselves mutagens, but are converted during metabolism in the liver to chemicals that are mutagens
What does sodium nitrite do?
Causes deamination of cytosine to produce uracil
This introduces a base pair substitution mutation
What is acridine orange
- Sandwiches itself between adjacent bases
- Distorts the DNA helix and causes insertions and deletion during replication
- Introduces a frameshift mutation
What were the three bacteria that we tested?
- E coli (trp-)
- Detects base pair
- S. Typhimurium A (his-)
- detects base pair
- S Typhimurium B (his-)
- Detect frameshift
Why did we use bacteria that could not synthesize certain amino acids?
Only bacteria that have undergone a reverse mutation will be able to synthesize the amino acid and grown on minimal medium
That is why we used trace amounts of amino acid, it is insufficient amount to allow auxotrophic bacteria to grow into colonies
Trick for pedigree
What are plasmids
non chromosomal supercoiled DNA
What are recombinant plasmids
replication of the recombinant plasmid in bacteria cells provides a means to produce many copies or cline the DNA of interest
What does TE buffer do?
eliminates proteins and other molecules found in the growth medium
What is SDS for in plasmid extraction for
(detergent) Lyses cells by disrupting lipids in the cell membrane
What is NaOH in plasmid extraction for
Denature proteins and DNA
What is potassium acetate for?
used to neutralize the base and potassium reacts with SDS to form an insoluble precipitate that traps proteins, chromosomal DNA, and membrane but not plasmid or RNA
What does low temperature do in plasmid isolation?
encourages precipitation
During the first centrifugation, what do you keep?
Separates proteins, chromosomal DNA and cell membrane (pellet) from plasmid DNA and RNA (supernatant)
What does use 95% ethyl alcohol do?
Precipitates plasmid DNA and RNA
What does the second centrifugation do?
Separates plasmid DNA and RNA (pellet_ from ethyl alcohol and inorganic salt (supernatant). Keep pellet
Why do we dry?
Ethyl alcohol will interfere with enzymatic digestion
What is RNase for?
Used to destroy RNA
What are the steps for DNA plasmid extraction
What is a restriction endonuclease?
An enzyme that makes a double-stranded cut within an intact DNA double helix at a specific nucleotide sequence called restriction site.
What is BamH1 do?
recognizes the six base pair palindromic sequence
- GGATCC
- CCTAGG
- G GATCC
- CCTAG G
What determines how rapidly DNA moves through gel matrix
Size/weight/shape of the DNA
Circular shape will migrate faster than linear
