W3/4 material Flashcards
Tagmentatoin - basics (step 3)
1. What cuts DNA to size and adds adaptors?
- Indices are added complimentary to what?
- What does PCR do? Are DNA fragments tagged now?
- Do magnetic beads bind varying sizes of DNA? under what conditions? what do we end up with?
- What do we do with th medium length fragments?
Bead transposases
Adaptors
Amplify and tagged DNA fragments.
Yes. Under different conditions. End result = medium length fragments
They are sequenced.
second step
what does the high salt solution do?
what do the cations attract?
what happens upon cation attraction?
what does the high salt solution keep on the column
what does ethanol do?
what elutes DNA from column?
breaks hydration shell, replacing water with positive charged ions
Negatively charged DNA
DNA adsorbed to silica membrane
DNA
get column dry
low salt buffer
QIAGEN DNA purification kit - to extract genomic DNA (first step)
What does the detergent (SDS) do?
what breaks the cell wall? how?
wat does the guanidine salt do?
disrupts/breaks the membrane via denaturing proteins
beads - breaks cell wall
breaks hydration shell via removing h2o from DNA + silica
step 4 - measure quantity + quality of DNA
what do we use? what machine?
what does it measure?
are proteins and DNA are absorbed at both 260 and 280?
a ratio of what means protein contamination?
spectrophotometry via nanodrop
absorbance of liquid
YES
ratio less than 1.8
Fluorometry (step 5)
what are you using?
how does this method work?
dye
only dye-binding DNA is measured
which is more accurate for measuring DNA concentration? Nanodrop or qubit? why?
Qubit. Bc it uses fluorescence for quantification
florescent dye binds only to dsDNA
Quantification of DNA using Qubit - fluorimetry
what instrument did we use?
How does it work?
What does it measure?
advantages? disadvantages?
qubit
measures using fluorescent dyes that specifically bind to dsDNA.
how much light is emitted (fluorescence intensity) measured, correlates w/ [DNA]
more accurate; does not measure purity.
what are the main bacterial shapes in cell morphology
- coccus
- bacillus - rod
what does a coccus shape look like?
what are the different types of cocci shaped bacteria?
circles.
Diplococci (2 cells), streptococci (chain of cells) , Staphylococci (ball of cells)
what does a bacillus shape look like?
what are the different types of bacilli shaped bacteria?
rod-shaped
diplobacilli, streptobacilli, cocobacillus (oval shaped)
Can you isolate a bacteria by spreading a broth culture on an agar plate? why?
no bc this method does not directly aim for isolation.
result in a uniform distribution of bacteria across the surface
If you wanted to cover an agar plate with a lawn of bacteria, which technique would be most appropriate? Why might you want to do this?
spreading method used for Antibiotic susceptibility testing, where you check the growth inhibition of a bacterial lawn in response to antibiotics
How do you prepare a spread plate?
serially dilute broth cultyure, add the broth culture to plate, add glass beads and swirl horizontally to cover the entire surface (or use a spreader), allow plate to absorb the liquid, incubate upside down
If you use the either method, do you need to dilute the culture first? Why or why not?
when using spread plates, you must serially dilute the culture first bc they are too dense
How do we store bacteria for a long time in the fridge?
How do we store bacteria for a long time in the freezer?
fridge - bac on a agar plate with parafilm around it or on an agar slant
freezer - prepare a glycerol (antifreeze) stock
what is a sarcina shape? tetrad? Palisades? hypha? stalk?
Sarcina → From Latin sarcina (“bundle” or “pack”), referring to the cube-like arrangement of bacteria.
Tetrad → From Greek tetras (τετράς), meaning “group of four,” describing bacteria arranged in fours.
Palisades → From Latin palus (“stake” or “post”), referring to the fence-like arrangement of bacterial cells.
Hypha → From Greek hyphē (ὑφή), meaning “web” or “weaving,” describing the thread-like structures of fungi or actinobacteria.
Stalk → From Old English stælcan (“to step, stalk”), referring to the elongated attachment structures in some bacteria.
Quantification of DNA + purity analysis - nanodrop ratio
Ratio < 1.8 indicates what type of contamination? why? does this increase or lower the ratio?
Protein contamination
Proteins absorb at 280 nm, so if there’s excess protein, A280 increases.
This lowers the A260/A280 ratio.
Ratio > 2.0 indicates what type of contamination? why? does this increase or lower the ratio?
Nucleotide contamination
Nucleotides absorb strongly at 260 nm so excess nucleotide increases 260
This increases the A260/A280 ratio.
Why do we “blank” (or “zero”) the instrument.
Define transmittance, absorbance, optical density.
Blanking removes background absorbance from the solvent, ensuring accurate nucleic acid measurement.
transmittance (T): fraction of light that passes through a sample compared to the incident light.
Absorbance (A): The amount of light absorbed by the sample
Optical Density (OD): used interchangeably w/ absorbance, measure of how much a sample absorbs light at a specific wavelength
What are the relationships between transmittance, absorbance, concentration, and dilution?
How can we determine the purity of our DNA sample? Why does this work?
As absorbance increases, transmittance decreases.
Higher concentration → higher absorbance
Dilution lowers concentration → lowers absorbance
By using the A260/A280 ratio
