MBG 3350 Term Test 1 Questions with Correct Answers Latest Update 2025/2026
Non-coding regions - Answers not all DNA encodes genes as some are not transcribed or
translated into proteins
Plasmids - Answers circular dsDNA that can replicate independently of the main chromosomes
of bacteria
Plasmids vs Chromosomal DNA - Answers - chromosomal DNA essential for life
- plasmids are dispensable as genes encoded within plasmids not usually essential for growth
under normal circumstances
Why are plasmids an advantage when under stress? - Answers Plasmids have resistance genes
Purpose of ORI? - Answers contains genes for DNA polymerase to replicate
Isolation and purification of plasmid DNA - Answers 1. break cell to release plasmid DNA
2. Separate plasmid DNA from chromosomal DNA
3. Remove RNA, proteins, lipids + small molecules
Isolation of Plasmid DNA (process) - Answers 1. Pelleting bacterial cells by centrifugation
2. Resuspend cells in solution with RNAse and metal chelator EDTA
3. Lyse cells with solution with alkaline pH and detergent
4. Precipitate chromosomal DNA and proteins with solution with acidic pH
5. Elute DNA from silica
Purpose of glucose in suspension solution - Answers Makes it isotonic and prevents cell lysis
before addition of NaOH/SDS
Purpose of NaOH/SDS in lysis solution - Answers - SDS has detergent properties that lyse cell
wall and membranes and denatures proteins
- NaOH breaks double helix
Purpose of Potassium acetate/acidic pH - Answers - causes chromosomal DNA to renature and
aggregate into insoluble mass
- chromosomal base pairs may not correctly rebind
- plasmid DNA renatures easily and is soluble
Purification of DNA by adsorption to silica resin - Answers 1. silica resin selectively binds
, negative charge on DNA in chaotropic salt solution
2. chaotropic ions remove water from DNA and allows plasmids to bind
3. wash resin with ethanol to remove RNA, carbs and proteins
Elution of DNA bound to silica resin - Answers elute plasmid DNA with sterile water or low salt
buffer solution
- water washes away Na+ from salt bridges and DNA will no longer bind
Spectrophotometric analysis of DNA - Answers - DNA and RNA absorb UV maximally at 260 nm
- dsDNA with concentration of 50 ug/mL has an A260 if 1.0
- RNA with concentration of 40 ug/mL has an A260 of 1.0
Spectrophotometric analysis calculation - Answers [DNA] = A260 x 50 x dilution factor
Purity of DNA - Answers 1.8-2.0 for pure DNA
- below 1.8 is protein contamination
- above 2.0 is RNA contamination
DNA examination by gel elctrophoresis - Answers 1. agarose is highly purified form of agar that
is readily soluble in polar solvents
2. agarose melted in buffered solution at high temp and solidifies into porous gel
3. DNA is negatively charged an migrates toward positive electrode
4. migration inversely proportional to log of size
5. DNA is stained by dye/chemicals to be observed
Relationship between [agarose] and DNA size - Answers low [agarose] used for larger DNA
pores
- pores are larger
Loading DNA sample onto agarose - Answers 1. Add small volume of DNA to 10x loading buffer
to a final concentration of 1x buffer to DNA sample
2. Load samples to wells with DNA markers
3. apply voltage across electrodes
4. stain gel to observe
Non-coding regions - Answers not all DNA encodes genes as some are not transcribed or
translated into proteins
Plasmids - Answers circular dsDNA that can replicate independently of the main chromosomes
of bacteria
Plasmids vs Chromosomal DNA - Answers - chromosomal DNA essential for life
- plasmids are dispensable as genes encoded within plasmids not usually essential for growth
under normal circumstances
Why are plasmids an advantage when under stress? - Answers Plasmids have resistance genes
Purpose of ORI? - Answers contains genes for DNA polymerase to replicate
Isolation and purification of plasmid DNA - Answers 1. break cell to release plasmid DNA
2. Separate plasmid DNA from chromosomal DNA
3. Remove RNA, proteins, lipids + small molecules
Isolation of Plasmid DNA (process) - Answers 1. Pelleting bacterial cells by centrifugation
2. Resuspend cells in solution with RNAse and metal chelator EDTA
3. Lyse cells with solution with alkaline pH and detergent
4. Precipitate chromosomal DNA and proteins with solution with acidic pH
5. Elute DNA from silica
Purpose of glucose in suspension solution - Answers Makes it isotonic and prevents cell lysis
before addition of NaOH/SDS
Purpose of NaOH/SDS in lysis solution - Answers - SDS has detergent properties that lyse cell
wall and membranes and denatures proteins
- NaOH breaks double helix
Purpose of Potassium acetate/acidic pH - Answers - causes chromosomal DNA to renature and
aggregate into insoluble mass
- chromosomal base pairs may not correctly rebind
- plasmid DNA renatures easily and is soluble
Purification of DNA by adsorption to silica resin - Answers 1. silica resin selectively binds
, negative charge on DNA in chaotropic salt solution
2. chaotropic ions remove water from DNA and allows plasmids to bind
3. wash resin with ethanol to remove RNA, carbs and proteins
Elution of DNA bound to silica resin - Answers elute plasmid DNA with sterile water or low salt
buffer solution
- water washes away Na+ from salt bridges and DNA will no longer bind
Spectrophotometric analysis of DNA - Answers - DNA and RNA absorb UV maximally at 260 nm
- dsDNA with concentration of 50 ug/mL has an A260 if 1.0
- RNA with concentration of 40 ug/mL has an A260 of 1.0
Spectrophotometric analysis calculation - Answers [DNA] = A260 x 50 x dilution factor
Purity of DNA - Answers 1.8-2.0 for pure DNA
- below 1.8 is protein contamination
- above 2.0 is RNA contamination
DNA examination by gel elctrophoresis - Answers 1. agarose is highly purified form of agar that
is readily soluble in polar solvents
2. agarose melted in buffered solution at high temp and solidifies into porous gel
3. DNA is negatively charged an migrates toward positive electrode
4. migration inversely proportional to log of size
5. DNA is stained by dye/chemicals to be observed
Relationship between [agarose] and DNA size - Answers low [agarose] used for larger DNA
pores
- pores are larger
Loading DNA sample onto agarose - Answers 1. Add small volume of DNA to 10x loading buffer
to a final concentration of 1x buffer to DNA sample
2. Load samples to wells with DNA markers
3. apply voltage across electrodes
4. stain gel to observe
