CHAPTER 1 (GENOMIC DNA EXTRACTION) Flashcards
(23 cards)
difference between genomic DNA and plasmid
genomic DNA is a linear DNA while plasmid is circular DNA
why do we need to extract DNA?
- DNA profiling
- disease diagnosis
- DNA sequencing
- cloning
- detection of pathogens
etc
purpose of cell lysis in DNA extraction
to break open cells and release the content
common methods of cell lysis?
- enzymatic lysis
- mechanical lysis
- heat/boiling
examples of enzymatic lysis
- lysozyme
- sds (detergent)
- EDTA (chelating agent)
- protease/proteinase
examples of mechanical lysis
- sonication - utilize high frequency. soundwaves to create tiny vibrations to burst open cells
- homogenizer - use blade/pressure
- grinders - manually break down the cell wall and membranes often with liquid nitrogen to make cells brittle
the time and temp of heat/boiling method in cells lysis
temp: 100 C
time: 10 mins
purose of DNA purification
to separate or purify DNA from proteins, cellular debris, and ther contaminants
common methods of DNA purification
- phenol/chloroform extraction
- spin column
ratios in phenol/chloroform extraction
1:1 of ohenol:chloroform
25:24:1 of phenol:chloroform:isoamyl alcohol
functions of phenol, chloroform, and isoamyl alcohol
phenol - denatures protein, form precipitates at interfacebetween aqueous and organic layer
chloroform: increases density of organic layer
isoamyl alcohol: prevents foaming
why spin column is preferred over phenol/choloroform method?
- faster, safer, easier.
- designed to work well with tricky samples, giving high quality DNA without messy and risky steps.
downsides of phenol/choloform
- time consuming
- toxic
- requires extractions (organic solvents) which means more steps and greater handling care
when to use phenol/chloroform method?
- wanting higher yields of DNA especially from tough complex samples.
- for large-scale DNA extraction (can be more cost-effective)
- can sometimes give exceptionally clean DNA particularly in difficult samples
in what condition does the binding occurs in spin column
in presence of high salt conc.
and is disrupted by elution with water
what do we use in DNA precipitation/recovery
inorganic solvent such as ethanol, isopropanol, etc + high salt conc. eg: sodium acetate.
what are the function of sodium acetate in DNA precipitation
DNA is negatively charged (because of its phosphate backbone).
Salt neutralizes these negative charges, so DNA strands don’t repel each other as much.
why do we have to precipitate DNA
we cant collect DNA from solution by just pullit it put bcs its dissolved. we have to form visible clumps (precipitate) to collect the DNA
what are the functions of ethanol in DNA precipitation
in alcohol, DNA becomes less soluble (DNA prefers water!).
So, DNA molecules clump together and fall out of solution (this is precipitation).
why DNA precipitation utilise high salt + alcohol
Salt helps DNA molecules come closer together.
Alcohol makes the solution bad for DNA to stay dissolved.
→ Together, they kick DNA out of the solution and you can collect it by centrifugation.
why get rid of carbohydrates in genomic DNA prep of plants
Carbohydrates (like polysaccharides) can contaminate your DNA sample when you extract genomic DNA from plants.
what happened if carbohydrates are not removed
They co-precipitate with the DNA (stick to it when you try to purify it).
They make the DNA sample sticky, viscous, and hard to pipette.
They inhibit downstream applications like PCR, cloning, or sequencing because enzymes (like polymerases) can’t work properly in contaminated samples.
They mess up the purity — your OD260/OD280 or OD260/OD230 ratios would be bad.
function of ctab
break open the cell membranes and remove carbohydrates by binding to the NA forming NA-CTAB complex
