Supplementary Introduction Flashcards
is the application of safety precautions that reduce a laboratorian’s risk of exposure to a potentially infectious microbe and limit contamination of the work environment and, ultimately, the community.
Biosafety
A core principle of biosafety is the _____________; therefore, organisms used in the teaching laboratory must remain in the laboratory and instructors must guard against inadvertent passage of the microbes out of the laboratory by a student or assistant.
containment of microorganisms
Potential Hazards in a Molecular Biology Laboratory
- What chemicals are being handled?
- Ethidium bromide (DNA stain)
- Chemicals and buffers for gel electrophoresis (agarose, polyacrylamide, SDS etc)
- High voltage during electrophoresis (~100-
200V) - UV light (viewing DNA on an agarose gel)
- Burns (burner, autoclave, chemical)
Molecular Biology Laboratory Cookbooks
(Classic): Ausubel et al. (eds)
(Classic): Sambrook and Russell
DNA Extraction Workflow
Genomic DNA Isolation:
- Sample preparation
- Cell lysis
- Protein removal
- DNA precipitation
- DNA rehydration
DNA Extraction Workflow
DNA Purification (Spin-Column Purification):
- Sample preparation
- DNA binding
- Wash
- DNA elution
I now have my DNA extract, what’s next?
Determine DNA Quality & Quantity
DNA Quantification equipment:
- UV-Vis spectrophotometer
- NanoDrop
- Qubit
Fundamental unit of double-stranded nucleic acids
Base pairs (bp)
“Sheared” DNA Extract
DNA fragments that have been broken down into smaller pieces during the extraction process, often due to mechanical or enzymatic forces.
He pioneered the use of 16S ribosomal RNA (rRNA) gene sequencing for phylogenetic studies
Carl Woese
30S subunit: 16S rRNA + 19 proteins
Prokaryotic ribosome
30S subunit
50S subunit
Agar sources:
Red Algae/Seaweed - Gelidium or Gracilaria
Nucleic acids (DNA and RNA) have a strong absorbance of UV light at 260 nm.
Many proteins and contaminants (like phenol) also absorb UV light, but at 280 nm.
Extract DNA using kitchen materials
Salts
Detergents
Surfactants
Why do we use agarose?
It is more translucent and clear
TBE buffer is less prone to overheating and is better for longer electrophoresis ruuns
Instead of ethidium bromide, we use
SYBR Safe, GelRed, or GelGreen
Diamond dye: yellowish light
SYBR dye: greenish light
Recommended Agarose Gel Percentages for Resolution of Linear DNA
Gel % DNA size range
0.5% = 1k - 30k bp
0.7% = 800 - 12k bp
1.0% = 500 - 10k bp
1.2% = 400 - 7k bp
1.5% = 200 - 3k bp
2.0% = 50 - 2k bp
DNA loading dye for 4k bp
Xylene cyanol FF
DNA loading dye for 500 bp
Bromophenol Blue
DNA loading dye for 50 bp
Orange G
common running buffer used in gel electrophoresis, particularly for separating nucleic acids (DNA and RNA) in agarose gels, providing the necessary ionic strength and pH conditions for optimal separation and preventing DNA degradation.
TAE buffer, or Tris-acetate-EDTA
What should be in your gel?
- “DNA ladder” or “MW marker”
- DNA samples
When you use more than 1 buffer in the gel
Flanking ladders
Ethidium bromide intercalates between base pairs
Intensity of the band is proportional to the concentration DNA.
Smearing – indicates DNA degradation!
Plasmid DNA Conformations
Relaxed circular form = low mobility
Linearized form = moderate mobility
Superhelical form = high mobility
Plasmid DNA should be predominantly supercoiled and free of genomic DNA and RNA
2 types of spectrophotometers
Analog
Digital
Spectrophotometer Cuvettes
Optical glass
ES Quartz
IR Quarts
PS or PMMA
A260/A280 = ~1.8 (Pure DNA)
A260/A280 = ~2.0 (Pure RNA)
Values < 1.8, indicates contamination probably caused by organic compounds or chaotropic agents, which absorb at 230 nm.
UV-Vis Spectrophotometry: DNA
Add DNA sample to molecular grade water in a cuvette. Read absorbance.
Sample is excited with filtered light (at the excitation wavelength, and the emitted light (at the emission wavelength) is recorded by a detector.
Qubit fluorometer
Why do we need to get the A320 reading?
the A320 reading is used to measure turbidity (cloudiness) of the sample, and is often subtracted from the A260 reading to get a more accurate DNA concentration measurement.
Calculation of DNA Yield
DNA yield (µg) = DNA concentration × total sample volume (ml)
DNA Concentration (µg/ml)
= (A260 reading – A320 reading) × dilution factor ×
50 µg/ml
Calculation of DNA Purity
DNA purity (A260/A280)
= (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)
