Lab final Flashcards
list 8 specimen types from which DNA can be isolated
whole blood, buffy coat, bone marrow, solid tissue, lavage fluids, bacteria/viruses, fungi, organelles (mitochondria)
what qualifies a specimen to be capable of DNA isolation?
any specimen that contains cells with nuclei
what are the parts of the anticoagulated blood specimen? where is the DNA found?
plasma, buffy coat, erythrocytes; DNA found in buffy coat
What is the purpose of the Chelex reagent in crude DNA extractions? Why is this important?
Chelex removes multivalent cations that can damage DNA.
Explain the purpose of each reagent in the strawberry DNA isolation procedure:
Shampoo: lysis the cells when the fruit is smashed in the buffer
Salt: promotes the precipitation of the DNA (removes DNA-bound proteins)
cold alcohol: DNA precipitates into the alcohol layer, DNA is insoluble in alcohol
What do the crude DNA extractions have in common? In what way do they vary?
Allow for release of DNA by cell lysis, the DNA is not purified
what is the difference bw extraction vs isolation?
isolation = DNA is cleaned up, isolated DNA is best
what do the letters stand for in PCI? what kind of a DNA isolation process is this?
phenol, chloroform, isoamyl alcohol; organic
what’s the first step in any DNA procedure? and what’s used?
lysis; detergents and proteases
what needs to be done if the pH needs to be lowered in an organic procedure? using what?
acidification; acetic acid and salt
in an organic isolation what is added to encourage precipitation (salts have already been added)
sodium acetate or ammonium acetate
what percentage of ethanol is added during ethanol precipitation? why is this done?
100%; DNA is insoluble in alcohol
what is the purpose of the 70% ethanol wash? what happens after this step?
dissolve salt with dilute alcohol; tube is inverted to air dry
what is the very last step of organic DNA isolation?
DNA is resuspended in buffer or water to dissolve DNA
List two reasons why the solid phase Qiagen spin column isolation method is better than the PCI method.
Highly purified DNA and less toxic chemicals
List two reasons why the PCI method is better than the Qiagen spin column isolation method.
molecular weight of DNA is higher; the amount and quality of DNA is higher
what are the 5 steps of the Qiagen method in order?
- Lysis using AL buffer and proteinase K
- incubation at 56 degrees
- addition of 100% ethanol
- addition of AW1/AW2 buffers
- Elution with AE buffer
what is the formula for calculating DNA concentration using spectrophotometry? How is the formula different for RNA calculations?
(A260)(50ug/mL)(dilution factor)
(A260)(40ug/mL)(dilution factor)
Explain why traditional spectrophotometry (not the Nanodrop) requires a DNA dilution step?
It is too concentrated to measure directly or in not in a volume that was measurable in a cuvette
at what wavelength does DNA, RNA, and protein absorb light?
260, 260, 280 nm
If the value for A280 is high, does that cause the purity ratio to be higher or lower?
lower
what is the ideal purity ratio for DNA? what is the range?
1.8; 1.6-2.1
what is an acceptable A260/A280 ratio for RNA?
1.7-2.3
An isolated DNA sample gives a A260/280 ratio of 1.4. What contaminant is likely present?
protein; ratio is low so that means a high 280 value
You isolate DNA and after spectrophotometry, you determine that the DNA has a concentration of 600 µg/mL and an A260/280 ratio of 1.3. What does this indicate about the quality of the DNA? Is the DNA conc accurate?
protein contamination; no, concentration is lower
Why is no dilution necessary for the Nanodrop?
When the arm is lowered and contacts the sample, it creates a liquid column with path length defined by gap bw the two optical surfaces
List three differences between Nanodrop spectrophotometry and fluorometry.
- Fluorometry is more sensitive than spectrophotometry
- Fluorometry is great for very small amounts of DNA
- Fluorometry involves the binding of a fluorescent dye to the DNA
what determines the intensity of the bands in a gel?
mass of DNA
what does it mean when a gel is smeared?
DNA is degraded
what indicates high quality DNA on a gel?
Intense, high molecular weight band with little to no smearing
In gel electrophoresis, why does DNA move toward the positive electrode? DNA is thus resolved based on what factor?
DNA is negatively charged; resolved size, molecular weight, # of base pairs
what are 4 characteristics of agarose vs polyacrylamide gel?
lower resolving power, very porous, better for separating large fragments, most commonly used, highest conc ~ 5
what are 4 characteristics of polyacrylamide vs agarose gel?
finer resolution, protein electrophoresis, toxic components, can go higher on concentration ~20
Which percentage agarose gel would be best to resolve two DNA fragments that are around 3000 bp in size?
1.5%, lower % for larger size
Which percentage polyacrylamide gel would be best to resolve two DNA fragments that around 20 bp in size?
20%; higher % for small fragments
Outline the steps for making an agarose gel, starting from agarose powder and 10X TBE.
- Determine the % of agarose concentration needed
- Dilute with buffer to proper concentration
- Heat solution to dissolve
- Cool to 55-60 degrees C
add gel red to see distance traveled - Pour gel into casting tray
- Allow to polymerize
- Remove comb, prepare to load
List the reagents/components used to make a polyacrylamide gel. Briefly state the purpose of each.
- Acrylamide powder – used to make stock solution
- Bis-acrylamide – used as a crosslinker
- Ammonium persulfate – initiates polymerization
- TEMED – stabilizes polymerization
what color is the negative electrode? where would this be connected?
black; side with the wells
List three purposes of loading dye for electrophoresis
Gives color to DNA for easier visualization
* Makes DNA denser so it sink to the bottom
* Has tracking dyes that separate during electrophoresis to indicate progress.
What is required to visualize DNA in a gel?
Adding gel red + UV light
