Microbiology quiz 18 Flashcards
dna extraction occurs in how many steps
3
what are the 3 badic steps for dna extraction
- Cell Lysis: a detergent (e.g., SDS) is used to
lyse cells and release cellular contents,
including DNA. - Denaturation: a heating step that denatures
protein and other cell components. - Purification: finally, the water-soluble DNA is
precipitated in cold alcohol as a whitish,
mucoid mass
Temperatures higher than __°C will denature DNA, and this is undesirable.
80
optimum wavelength for
absorption by DNA.
260
absorbance of A260nm of 1 corresponds to ____ μg/ mL of double-stranded DNA
(dsDNA). T
50
Reading absorbance at _____ nm and calculating the following ratio provides an estimate of the
sample’s purity
280
f the sample is reasonably pure nucleic acid, the ratio will be about
1:8
lower then a reading of 1.6 means
higher then a reading of 2.0 means
1.6- contaminated
2.0- needs to be diluted further
exctract dna from
e.coli
Spin the 5 mL sample in the tabletop _______ slowly for 10 minutes to produce a cell pellet
centrifuge
what do you add to e.coli
Add 200μL of 10% SDS to the E.coli.
Add 30 μL of 20 mg/mL Proteinase K solution, or “half a pinch” of meat tenderizer
When cooled, squirt 400μL of cold 95% _____ into the preparation
isopronal
Spectrophotometry can be used in both qualitative and quantitative
analysis of
DNA, RNA, and Proteins
Importance of Heating the Cell Lysate
helps break open cells releasing the DNA into the solution
Why does the extraction work better with cold 95% isopropanol than
with room-temperature 95% isopropanol?
Cold isopropanol promotes DNA precipitation by decreasing its solubility in the solution
beer lambert equation
c=A/eL
L is light path
e is wavelgnth
A is uv absorbance
Nucleic acids absorb light with a peak at_____ nm.
Purified proteins absorb light at a peak
of ____ nm.
Many contaminants left over from
extraction protocols absorb at either __ nm or ___ nm
230; 280;
280;230
pay close attention to the
Pay close attention to the graduations on the pipette
The 100 μl and 250 μl and 1 ml marks will be used
as units of measurement throughout the molecular
labs
DNA can be quantified by:
cutting the DNA with a restriction enzyme,
running it on an agarose gel,
staining gel,
comparing the intensity of the DNA with a DNA marker of known concentration
quantified DNA can be isolated and examined further using
PCR
analysis
