bio lab final Flashcards
ph<pI
positively charged
ph>pI
negatively charged
how many times do you wash after seconary antibody
2
positive control
contains variable being tested for, demonstrates positive result, confirms everything is running correctly
negative control
does not contain tested variable, confirms no false positives, demonstrates negative results
Reusing a pipet tip when transferring controls and samples from tubes to wells
is more likely to cause false positives than false negatives.
Which of the following errors would result in a negative result for all samples in an ELISA (including positive controls)? Select all that apply.
Failure to add antigen.
Correct
Failure to add funtional primary antibody.
Correct
Failure to add functional secondary antibody.
Correct
Failure to add functional substrate.
Which of the following errors would result in positive results for all samples in an ELISA assay (including negative controls)?
Failure to perform the wash step after incubation with secondary antibody.
primase
makes first few nucleotides
steps of pcr
denaturing at 90-95, annealing at 55, extension at 72
how many genes in mt dna?
37, 13 protein, others for trna or rrna.
agarose concentration
0.5%>10 kb
1% for 500-10 kb
2% for smaller than 1 kb
chelex
removes metal ions that may catalyze dna digestion
mg2+ function
cofactor, facilitates nucleotide phosphate bonds between primer and incoming nucleotides
components of pcr rxn
dna, nucleotides, buffer with mg2+, primers, dna polymerase
steps for a pcr reaction
incubation with cacl2, cells, and the plasmid– cacl2 makes the cells competent
heat shock to stress cells and destabilize membranes
recovery– incubation for gene expression
arabinose function
binds to protein called arac on pbad so it allows binding of rna polymerase
griffith
found that combining smooth killed pneumococci with nonvirulent rough pneumococci caused death. transforming principle present
avery, macleod, mccarty
s-strain cells fractionated into classes of molecules and only dna transformed r strain into s strain
cells can be made permeable to dna using
calcium chloride, heat shock, electroporation, protoplast formation, microprojectiles
components of plasmid
origin of replication, gene of interest, genes with selective advantage, and polylinker area with multiple endonuclease cleavage areas
successful transformation needs
host cells, plasmid with selectable marker
constitutive gene
constantly expressed
inducible
gene only in certain conditions
total amount of pglo used
volume*concentration
fraction of dna
volume spread/ total volume (cacl2+lb broth+plasmid dna)
micrograms spread
amount of dna*fraction
transformation efficiency
colonies/micrograms spread
plasmid pglo characteristics
Plasmid: pGLO - selectable marker (bla -
ampicillin resistance gene), gene for GFP,
origin of replication (ori), polylinker area,
special gene regulation system (Promotor -
P(BAD) and araC gene)
beer’s law
a=bec (b=1 cm pathlength, c-concentration)
kmno4 and chemicals
plastic cuvettes
proteins
quartz cuvettes and plate readers
nucleic acids
low volume spectrophotometer
ph of acid phosphatase reaction
4.5
nitrophenyl phosphate to nitrophenol and phosphate
acidic amino acids
aspartic acid, glutamic acid
basic amino asids
lysine, arginine, histidine
At what temperature is Taq DNA polymerase most active?
72-75
The two essential components that need to be added in addition to the “Dream Taq Green PCR Master Mix” to set up the PCR reaction are:
Primers and DNA template
The two primers for one PCR reaction should be complementary to each other.
F
The direction of synthesis of the new DNA strand in each cycle of the PCR is from 5’ to 3’ end.
T
Which of the following are true in regards to mitochondrial DNA (mtDNA)? Choose all that apply.
The mitochondrial genome contains 37 genes.
Correct
There are more copies of mt genes than nuclear genes per cell.
Makes cells permeable to plasmid DNA
Correct C.
CaCl2
Correct C.
CaCl2
Selects for the growth of transformed cells
Correct E.
Ampicillin
Correct E.
Ampicillin
Provides the GFP gene
Correct B.
pGLO plasmid
Correct B.
pGLO plasmid
Induces GFP gene expression
Correct A.
Arabinose
Correct A.
Arabinose
To increase the efficiency of transformation, cells will be placed in a 42°C water bath for 50 seconds during which step of the procedure?
heat shock
Which of the following is a method for making cells competent (permeable to plasmid DNA) ?
electroporation, microprojectile, protoplast formation
In our experiment, expression of the beta-lactamase gene (from the plasmid) is constitutive.
T
HB101 is a strain of E. coli that is naturally antibiotic resistant.
F
During a transformation experiment with pGLO, the gene for GFP will remain turned off if arabinose is present in the media.
F
Which of the following wavelength ranges describes visible light?
400-700
x axis absorption and standard curve
wavelength, concentration
We will discover the lambda max (λmax ) of a chemical solution by generating and analyzing the absorption spectrum.
t– accuracy and sensitivity
The molar absorptivity of a chemical (ε) is always constant at any wavelength (ie – it would be the same at 300nm and 400nm and 500nm)
F
Autozero (pressing Measure Blank) is required when a new wavelength is set
T
The initial velocity of an enzyme catalyzed reaction is determined by
calculating the amount of product formed over time while the amount of product is increasing in a linear manner
Which of the following influence the rates of enzyme-catalyzed reactions?
:
Correct
Concentration of substrate
Correct
Presence of activators or inhibitors
Correct
Temperature and pH
Correct
Amount of enzyme
The experiment you will be performing in lab assays the reaction rate of wheat germ acid phosphatase at a temperature of [a]°C.
37
Question Correct Match Selected Match
Nitrophenyl phosphate
Correct C.
substrate
Correct C.
substrate
Nitrophenol
Correct A.
product
Correct A.
product
Acid phosphatase
Correct D.
enzyme
Correct D.
enzyme
1.5% KOH
Correct B.
stops reaction and allows absorbance of product to be measured
Correct B.
stops reaction and allows absorbance of product to be measured
nitrophenol standard curve
x=cocnentration
y=absorbance
The isoelectric point (pI) of an amino acid is defined as the pH at which the amino acid does not migrate in an electric field or the pH at which an amino acid bears no net charge.
T
Which of the following four proteins in Part A of today’s experiment is the most acidic protein?
bovine albumin
1 out of 1 points
Correct
If amino acid Lysine with a pI of 9.7 is dissolved in a gel running buffer of pH 6; at this pH, Lysine is a___________ and it will migrate toward____________ during the native agarose gel electrophoresis
cation, cathode
If your group is assigned to prepare 60 ml of 1.2% agarose gel solution for part B, what materials are required to prepare the gel solution. Select the amount of agarose and the amount of the correct buffer needed.
Correct
0.72 g of agarose
Correct
60 ml of electrophoresis buffer with pH at 9.2
Which two of the following statements about the separation of molecules are true?
Separation of Protein using “Native” or “non-denaturing” gel electrophoresis is on the basis of the net charges
Correct
Separation of DNA molecules using gel electrophoresis is on the basis of sizes
What is the basis of separation of the HbA and HbS in Part B of today’s experiment?
charge
Incubation: the cells mixed with a transformation solution of CaCl2 (calcium chloride) and the plasmid.
* Heatshock: the cells and DNA mixture are then subjected to a treatment at 42ºC for 50 seconds; the temperature changes destabilize cell membranes and create thermal imbalance.
* Recovery: a short incubation period to begin expressing their newly acquired genes
