Term
| International System of Units |
|
Definition
Mass:Kilogram (kg)
Amount of substance: Mole (mol) |
|
|
Term
|
Definition
Mega: 10^6
Kilo: 10^3
Milli: 10^-3
Micro: 10^-6
Nano: 10^-9
Pico: 10^-12 |
|
|
Term
|
Definition
| L > ml > microL > nL > pL |
|
|
Term
PDF 2: Mole
6.023 x 1023 molecules of the substance (Avogadro’s number). |
|
Definition
-Measurement of mass of a substance
-The number of grams in 1 mole of the substance is equal to its molecular weight. |
|
|
Term
|
Definition
The same as the relative molecular mass of a molecule expressed in Daltons.
The molecular weight is expressed in
grams/mole. |
|
|
Term
PDF 2: Molar solution
(Molarity is abbreviated M.) |
|
Definition
A molar solution of a substance is one mole of that substance in a litre of solution.
The concentration of ions. |
|
|
Term
PDF 2: Percent solutions
-Gram Percent Solution |
|
Definition
-Deals with solutes
Expressed as weight/volume ratio.
1%(w/v) solution contains 1g of the
solute per 100ml of solution. |
|
|
Term
PDF 2: Percent solutions
-Gram Percent Solution |
|
Definition
-Used with liquids.
-Indicates the number of millilitres of
solute dissolved in 100ml of solution. |
|
|
Term
| Metric System Conversations |
|
Definition
1μl = 10^(‐6) litres
1,000,000μl = 1 litre
1ml= 10^(‐3) litres
1, 000ml= 1 litre |
|
|
Term
PDF 2: pH
-Power of the hydrogen ion in solution |
|
Definition
-pH=‐log [H+].
-Based on dissociation constant of water.
-pH scale: 0 to 14
- Kw=[H+] x [OH‐] |
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
PDF 2: pH
-acid dissociation |
|
Definition
Kw=[H+] x [OH‐]
-25°C, pure water dissociates until the acid and base concentrations are equal at 10‐7 molar and thus the pH of pure water is 7.
In aqueous solutions at 25°C, the
product of the [H+] and [OH‐] are always constant at 1 x 10‐14. Thus, if one introduces an acid or a base to the system, the pH will drop or rise in a manner proportional to the amount of acid or base added. |
|
|
Term
|
Definition
Kw= [H+] x [OH‐]
[H+] = Kw/ [OH‐].
[NaOH]=0.01M = 10^(‐2)M
[OH‐]=10^(‐2).
[H+]= 10^(‐14)/10^(‐2)
[H+]=10‐12
pH=‐log[H+]
pH=‐log(10^(‐12))
pH=12. |
|
|
Term
PDF 2: pH
-How does one measure pH? |
|
Definition
Protons are charged molecules that establish an electrical potential.
pH meter measures the voltage between two electrodes.
One of the electrodes is usually a special type of borosilicate glass, which is selectively permeable to H+, but not other ions.
It is a pH‐dependent electrode.
When placed in a solution, the H+ will flow from the side where they are more concentrated to the side of lower concentration. This in effect establishes an electrical potential across the glass, the magnitude of which is expressed by a equation |
|
|
Term
PDF 2: pH
-How does one measure pH: Equation to measure potential |
|
Definition
E= 2.303(RT/F)Log([H+sub1]/[H+sub2]
E= electrical potential
R=gas constant (derived from PV=nRT)
T=absolute temperature
F=Faraday constant (charge per gram equivalent weight of an ion).
H+sub1=molar concentration of H+ on inside of glass electrode
H+sub2=molar concentration of H+ on outside of glass electrode |
|
|
Term
PDF 2: pH
-How does one measure pH: General Info |
|
Definition
If the H+ concentration of one of the solutions is fixed, the potential will be proportional to the pH of the other solution.
In order for electrical current to flow in a system, there must be
a closed the circuit.
In the case of the pH meter, a reference
electrode is employed both to close the circuit and to provide a stable reference potential which is pH‐independent.
The reference electrode is typically filled with Ag/AgCl2 and contact with the solution to be measured is through a fiber or capillary that is connected by a solution of saturated KCl. A small amount of KCl flows out into the sample to be measured (usually less than 10ul/hr), and this effectively makes electrical contact with the solution. |
|
|
Term
PDF 2: pH
-How does one measure pH: General Info II |
|
Definition
-pH meter is capable of detecting very small changes in electrical potential.
Measuring the electrical potential of known standards, and temperature, pH meters calculate the slope of an
electrode system.
This is a constant that relates electrode potential to the logarithm
of the concentration.
The efficiency of the electrode is the actual electrode slope divided by the theoretical slope.
In order to standardize the instrument,
the slope must be determined, which means that at least two different standard buffers should be used. As a practical limitation, the pH of the sample you want to measure should fall between the two standards. Some of the more modern meters are microprocessor controlled and typical measurements can be made accurately by using only one standard and accepting the slope calculated from the last two point
standardization. |
|
|
Term
PDF 2: pH
-How does one measure pH: General Info III |
|
Definition
| -electrode bulb must remain wet |
|
|
Term
PDF 2: Biological buffers & practical considerations for adjusting pH
-Buffer |
|
Definition
A solution containing a mixture of a weak acid and its conjugate weak base.
-Capable of resisting substantial changes in pH upon the addition of small amounts of acidic or basic substances. |
|
|
Term
PDF 2: Biological buffers & practical considerations for adjusting pH
-Biological Buffers |
|
Definition
Buffering required to maintain the integrity and function of macromolecules outside the cell
(in vitro).
The quality of a buffer depends on its buffering capacity; that is, the capacity to tolerate the addition of acid or base. The pH of a solution of weak acid or
base can be calculated from the Henderson Hasselbach equation |
|
|
Term
PDF 2: Biological buffers & practical considerations for adjusting pH
-Henderson Hasselbach |
|
Definition
pH=pKa+ [A-]/[HA]
pKA=the centre of a weak acid base buffer region
A- = Basic species
HA = Acid species |
|
|
Term
PDF 2: Biological buffers & practical considerations for adjusting pH
-pKa |
|
Definition
-pKa of a buffer is the pH value at which the concentrations of the
basic and the acidic species are equal
-Also equal to the negative logarithm of Ka).
-By knowing the pKa of the buffer involved and the concentrations of the proton donors and acceptors, can calculate the pH of a solution.
Similarly, the relationship can be used to determine the ratio of proton donor to acceptor to be used to make a solution at a specified pH. |
|
|
Term
PDF 2: Biological buffers & practical considerations for adjusting pH
-dissociation of a weak acid |
|
Definition
equilibrium constant for this reaction is called the acid dissociation constant,
Ka, and it is expressed as:
Ka=([H+][A-])/[HA]
[H+]= proton concentration
[A-]= concentration of basic species
[HA] = concentration of acid |
|
|
Term
PDF 2: Biological buffers & practical considerations for adjusting pH
-problem |
|
Definition
| Fuck you, look at the lab manual |
|
|
Term
PDF 2: Biological buffers & practical considerations for adjusting pH
-How is pH adjusted? |
|
Definition
-All dry (and liquid) chemicals are dissolved into the solvent (usually DIW).
-Standardized pH meter to the appropriate value.
-rinse the electrode well with DIW over the waste beaker, then immerse the tip into your solution.
-Let the electrode equilibrate for about 10 seconds.
-Let pH value on the meter should stabilize.
-With gentle stirring, measure the pH.
-If above the desired value, add acid.
-If below the desired value, add base.
If the solution is strongly buffered add a strong acid (i.e. 5N HCl) or base (i.e. 5M NaOH).
Why: minimizes volume changes and quickly leads to adjustment to the “ballpark” pH.
If your solution is weakly buffered or near its desired pH such that only a small adjustment is needed, use a weak acid (0.1M HCl) or base (0.1M NaOH).
Add the acid or base drop‐wise and let the solution equilibrate after each drop.
Stabilize pH meter. |
|
|
Term
PDF 2: Biological buffers & practical considerations for adjusting pH
-clean up |
|
Definition
When at desired pH finished with the exception of cleaning up and properly taking care of the electrode and pH meter.
* Be sure the standard solution has not been contaminated, and close the
container (may be in a beaker which needs to be parafilmed)
* Be sure that you have rinsed the electrode well with the DIW, and
immersed it in the storage solution.
* Be sure to turn off the pH meter or set it to standby mode.
* Wipe up any spills and take care that the acids and bases used are
properly stored and the containers capped. |
|
|
Term
|
Definition
-tryptone (a mixture of partially hydrolyzed
proteins)
-yeast extract (mushed up yeast cells)
-sodium chloride |
|
|
Term
|
Definition
-Lamp or light source is
directed through a monochromator, which can be set to different wavelengths.
-Filter out the entire light spectrum with the exception of the desired wavelength.
Light passes through the sample [or the reference standard] in the cuvette holder that can be moved with the slide rod (other spectrophotometers have a rotating wheel inside) that permits one to measure the absorbance of the standard and that of the samples positioned in cuvettes around
the wheel, and is collected by a phototube.
The phototube is connected to a metering device, which records the output. |
|
|
Term
|
Definition
Need to add a significant
amount of NaOH to this in order for the EDTA to dissolve |
|
|
Term
PDF 4: Incidence Light
-Standard |
|
Definition
| In practice, the incident light coming from the monochromator is passed through the standard and the transmitted light is received by the phototube. This value is set to 100% light transmission, or 0% absorbance |
|
|
Term
PDF 4: Incidence Light
-Sample |
|
Definition
| The value of the transmitted light is then compared to that of the standard or a blank (a blank is a solution which has the same composition as that being measured but in general no macromolecules are present). The decrease in light transmission (or increase in absorbance is related to the concentration of the sample. |
|
|
Term
|
Definition
absorbance is directly proportional to
solute concentration (c) and to the length of the light path (l)
A = αcl
α ‐ is the absorption coefficient
c ‐ is the concentration
l ‐ is the light pathlength
-In most cases the absorption coefficient α can be found in the literature, or it can be
experimentally determined. This is done by plotting a standard curve of absorbance
vs. concentration of the pure compound, and then determining the slope of the line. |
|
|
Term
| PDF 4: Transmittance and Absorbance: |
|
Definition
-transmittance and absorbance are related to one another in a
linear (inverse log) fashion. |
|
|
Term
PDF 4: Transmittance and Absorbance
-Transmittance equation |
|
Definition
T = (I/IsubO)
IsubO = intensity of the light striking the sample
I is the intensity of light
after passing through the sample.
-transmittance is expressed as percent
transmittance |
|
|
Term
PDF 4: Transmittance and Absorbance
-percent transmittance |
|
Definition
|
|
Term
PDF 4: Transmittance and Absorbance
-Absorbance |
|
Definition
Absorbance is also known as the optical density of the sample, and it is a
logarithmic function of transmittance. |
|
|
Term
PDF 4: Transmittance and Absorbance
-Transmittance |
|
Definition
| -Transmittance is the fraction of incident light that passes through the sample (is transmitted). |
|
|
Term
PDF 4: Transmittance and Absorbance
-Absorbance equation |
|
Definition
A = log (1/T)
A = log (IsubO/I) |
|
|
Term
| PDF 4: Plotting Data and Extinction Coefficient. |
|
Definition
When spectral data is plotted, it is usually the absorbance that is plotted against the concentration or wavelength. In most cases, a standard light path of 1cm is used (i.e. 1cm cuvettes are used) and the absorbtivity parameter is known as the extinction coefficient. Thus, if the extinction coefficient is known, the concentration
of a pure substance in solution can be calculated from its absorbance. |
|
|
Term
| The electromagnetic radiation spectrum: |
|
Definition
In general most biological macromolecules absorb light in the visible or
UV range, and colorimetric tests utilize dyes that can be measured in the visible
range. |
|
|
Term
PDF 4: Assays to determine protein concentration
Why do Proteins absorb in the ultraviolet range? |
|
Definition
They (usually) have aromatic amino acids in their sequences (tyrosine and tryptophan for example) and these absorb light in the 280nm range.
However, unless the sample is
a pure protein and the extinction coefficient is known (both of which are usually not
the case), it is difficult to equate absorbance with concentration except very roughly
(Abs280 of 1 usually represents 0.4‐1.5mg/ml of protein).
The actual value depends on the amino acid composition of the protein.
Absorbance at 280nm only gives a rough value for concentration.
An absorbance peak at 205nm can be used as a more reliable indicator of concentration.
Peptide bonds absorb at this wavelength, and this results in a more
accurate answer, which is much less biased by amino acid composition.
@ 205nm in the far UV range, oxygen begins to absorb and many commonly
used buffer salts also give high backgrounds. |
|
|
Term
PDF 4: Assays to determine protein concentration
-benefits to absorbance |
|
Definition
Although there are drawbacks to using
absorbance to quantify a sample, it is a non‐destructive method, and the sample being measured can be returned to its tube for further characterization. |
|
|
Term
PDF 4: Assays to determine protein concentration
-ploting absorbance |
|
Definition
-plotted with the
concentration on the abscissa (horizontal axis) and the absorbance or
transmittance value on the ordinate (vertical axis). These are your standard curves. |
|
|
Term
|
Definition
-prokaryotic
-live almost everywhere (frozen tundra to the hot acid springs of Yellowstone Park where they live at pH 2 and at nearly 100°C.)
-Most live in more pleasant environments, such as the human digestive system. |
|
|
Term
| Most commonly used bacteria in lab? |
|
Definition
|
|
Term
Most commonly used bacteria in lab?
why? |
|
Definition
-Most common host for the propagation of recombinant DNA molecules.
Many strains of E. coli can grow on a carbon source (like glucose) in simple inorganic salts. |
|
|
Term
|
Definition
-E. coli is grown in a medium known as LB (Luria Broth or Luria‐Bertani broth).
Other factors:
oxygen (for complete respiration to occur)
-temperature.
Culture oxygenated by rapid shaking in an incubator
E. coli optimal growth temperature at 37°C.
At optimal reproduce in about 20 minutes. |
|
|
Term
|
Definition
Place media in autoclave
-Autoclave:high temperatures (around 121°C) and relatively high pressures (around 15 lbs/in2).
Treatment of the media, reagents, or
laboratory apparatus for 20 minutes to kill all microorganisms, and render the materials sterile. |
|
|
Term
|
Definition
Sterile media and
reagents permit you to culture bacteria of your choice without contamination by
other microorganisms. |
|
|
Term
| Why might something explode in the autoclave? |
|
Definition
-hotter than boiling water, and infrequently, superheated bottles explode when bumped.
-necessary to keep all screw caps loose during autoclaving, and to handle all hot materials with extreme caution. Let the materials sit for a short time to cool before handling them |
|
|
Term
| Why grow bacteria in the pressence of antibiotics |
|
Definition
Autoclaving the antibiotics will destroy the activity of the drug,
so they must be added after autoclaving and after the medium has cooled to <60°C.
The stock solution of ampicillin has been filter sterilized (to avoid contamination)
and is made as a 1000X stock. |
|
|
Term
| Antibiotics: Bacteriostatics |
|
Definition
do not kill the organism, but simply make it
incapable of reproduction (in bacteria growth equals cell division).
Tetracycline |
|
|
Term
| Antibiotics: bacteriocidal |
|
Definition
Most bacteriocidal antibiotics inhibit cell wall synthesis
Ampicillin, a derivative of penicillin |
|
|
Term
|
Definition
antibiotic interferes with the synthesis of the peptidoglycan
layer between the inner and outer membranes. It kills cells attempting to divide or cells laying down a new peptidoglycan layer.
The plasmids used have a resistance to ampicillin (similar in structure to penicillin) by virtue of the fact that
they contain a bacterial β‐lactamase gene. |
|
|
Term
|
Definition
The β‐lactamase cleaves the ampicillin
molecule such that it can no longer affect peptidoglycan synthesis, and therefore the
cell can make a cell wall and will reproduce. |
|
|
Term
|
Definition
antibiotic binds to the 30S subunit of the bacterial ribosome and interferes with the
binding of aminoacyl tRNAs to the A site, inhibiting translation. Consequently, the
cells cannot make new proteins. |
|
|
Term
PDF 5: Growth and Enumeration of Bacteria
Doubling time of E. coli |
|
Definition
| There are many strains of bacteria, and healthy cultures of E.coli have a doubling time (or generation time: the time required to reproduce to double the number of cells) of about 20 minutes in rich medium at 37°C. |
|
|
Term
PDF 5: Growth and Enumeration of Bacteria
-Turbidity (optical density): |
|
Definition
Turbidity measurements assess the growth of cultures.
-a measure of the number of particles (bacteria) in a suspension.
densely populated cultures will be very turbid (cloudy) and give high turbidity values.
Spectrophotometer: measured by light
transmittance or by absorbance.
high values could be due to a very healthy culture, which is exponentially growing, or it may be due to
a culture, which has been depleted of nutrients and may consist of more dead cells than live ones.
see graph page 6 |
|
|
Term
PDF 5: Growth and Enumeration of Bacteria
-Turbidity (optical density): Graph
Log CFU/ml vs. time |
|
Definition
Four parts:
lag
logarithmic
stationary
death |
|
|
Term
PDF 5: Growth and Enumeration of Bacteria
-Turbidity (optical density): Graph
-lag |
|
Definition
| represents the time shortly after inoculation, in which the cells are recovering and adjusting their metabolism to most efficiently utilize the components of the medium. In lag phase, there is little cell division. |
|
|
Term
PDF 5: Growth and Enumeration of Bacteria
-Turbidity (optical density): Graph
-logarithmic |
|
Definition
| Now the cells are beginning to divide quickly which is called the exponential growth or logarithmic phase. In 'log' phase, the cells reach their maximum rate of reproduction. During this time one can take aliquots of the cells and perform cell counts at short intervals and a plot of the cell number versus time will produce a straight line. |
|
|
Term
PDF 5: Growth and Enumeration of Bacteria
-Turbidity (optical density): Graph
-stationary |
|
Definition
In stationary phase, the population is very large, toxins begin to accumulate,
nutrients are depleted, the pH of the culture drops, etc., with the result being that cells slow their rate of reproduction (cell growth equals cell death). |
|
|
Term
PDF 5: Growth and Enumeration of Bacteria
-Turbidity (optical density): Graph
-death |
|
Definition
| Death phase can also occur exponentially as the culture conditions are unable to support life. |
|
|
Term
PDF 5: Growth and Enumeration of Bacteria
-Colony Count |
|
Definition
In a logarithmically growing culture, the number of bacteria is in the high 10^(7)/ml to low 10^(8)/ml range.
Even if you were to plate 1μl (which cannot be very accurately measured), you would theoretically get 10^(4) colonies on the plate. In addition to being tedious to count, this number would result in the touching of
adjacent colonies, and a smooth ‘lawn’ of bacteria would be formed. What is
typically done is to take an aliquot of the culture and perform a series of dilutions in nutrient medium. One starts with a relatively large volume (1 to 10ml) that can be accurately measured. Remember you are pipeting bacterial cells and sterile technique is a must.
see page 7 |
|
|
Term
|
Definition
The ability to
propagate individual DNA molecules |
|
|
Term
| PDF 6: Restriction enzymes |
|
Definition
-Enzymes isolated from prokaryotes
-recognize specific DNA sequences
-Act as endonucleases
-Break the phosphodiester backbone of DNA at specific sites. |
|
|
Term
PDF 6: Restriction enzymes
-Type II |
|
Definition
| -Restriction enzymes that recognize palindromic sequences. |
|
|
Term
|
Definition
-Palindromes are regions of two fold symmetry
-nucleotide sequence is identical on both strands if read from the same direction.
eg:
5’ CCTGTCAGAATTGTAAGCTTCAATGGCGCATATCG3’
3’ GGACAGTCTTAACATTCGAAGTTACCGCGTATAGC5’ |
|
|
Term
|
Definition
A collection of clones
-represents either the entire genome of an organism or the genes that are expressed as mRNA |
|
|
Term
PDF 6: Library
-cDNA Library |
|
Definition
| -target DNA is generated by making a DNA copy of mRNA from the organism (or tissue or developmental stage) of interest. |
|
|
Term
PDF 6: Library
-genomic library |
|
Definition
| -target DNA is generated (usually) by restriction enzyme digestion of genomic DNA. |
|
|
Term
|
Definition
-plasmid or a bacteriophage molecule
-Three distinct properties:
• an origin of DNA replication such that the vector can replicate autonomously.
• single restriction enzyme sites for the introduction of the target DNA.
• a selectable marker gene (usually one conferring antibiotic resistance). |
|
|
Term
|
Definition
| -bacterium (or sometimes a yeast strain), which is used to propagate individual DNA molecules. |
|
|
Term
| PDF 6: Target or insert DNA |
|
Definition
| The foreign DNA fragment inserted into a vector |
|
|
Term
| PDF 6: Multiple cloning site: |
|
Definition
| A region of a vector which contains several unique restriction enzyme recognition sites which may be used to clone target sequences. |
|
|
Term
| PDF 6: How to Make Cells competant for transformation |
|
Definition
Use CaCL2
-Ca(+) ions interact with the negatively charged phosphates in the phospholipid molecules of the E. coli cell membrane.
At low temperature, this creates a relatively rigid membrane structure that has the propensity to form
minute pores where the positive charges of the calcium shield the negative charges of the phospholipids.
-Since DNA also has a phosphate backbone, it interacts with the CaCl2 and forms a complex that precipitates onto the cells.
-After a short time, a heat shock is given which is thought to generate transient holes in the membrane and permit the plasmid DNA to ‘rush in’.
-Now the plasmid is inside the cell. If the plasmid confers some new and
advantageous property to the bacterium, these cells may then be able to grow under conditions that would not ordinarily allow for growth. |
|
|
Term
|
Definition
-Grow bacteria to mid log phase
-Harvested, resuspended in a small amount of sterile, ice‐cold 50mM CaCl2
-leave on ice for about 20 minutes, harvested, resuspended in fresh CaCl2
-Increase efficiency of transformation by leaving cells over night in refrigerator. |
|
|
Term
| How much does overnight "seasoning" of cells increase efficiency of transformation? |
|
Definition
5 to 10 fold
Why?
-Such high efficiency competent cells need because small amount amount of DNA available or want to obtain as many clones as possible (building a library).
-Under optimal conditions such cells may give rise to over 107 transformants per microgram of supercoiled DNA. |
|
|
Term
| How are libraries generated? |
|
Definition
Consider a genomic library in bacteriophage lambda (λ).
The genomic clone selected may contain as much as 23kbp of insert DNA, yet very
little of this may be relevant to the ultimate goal (for example, the promoter of interest may be about 1kbp of the total).
Take the long λ clone and make smaller subclones from it.
For this purpose, there is generally a lot of DNA available, and its complexity is low (that is, there are many copies of a small number of different fragments present).
In practice, this means that the transformation efficiency does not need to be high for typical subcloning purposes. |
|
|
Term
|
Definition
-typically small molecules, usually not more than 10kbp in length
-Plasmids found in E. coli
as supercoiled molecules, but during the purification process, nicking can occur. |
|
|
Term
|
Definition
| Replication in most E. coli strains generates molecules that are supercoiled |
|
|
Term
|
Definition
-the topology of the molecule
-described by a solenoid or coiled coil.
-superhelical tension gives the molecule a smaller surface area than
a circular, non‐supercoiled or linear molecule. |
|
|
Term
|
Definition
-occurs by physical forces or can be due to the action of nucleases
(endonuclease activity), with the result being the relaxation of superhelical density (for one end of the double helix is now free to rotate) and the formation of open circular DNA |
|
|
Term
| How does different DNA topology look on gel? |
|
Definition
See PDF 6 page 5
Forms are all the same size, but their conformation dictates how effectively they can move through a gel matrix.
Supercoiled: Is very compact and rapidly
moves through the gel matrix
Open circle: Has greatest surface area and is least flexible.
Linear: can easily pass through gel matrix but may become tangled.
Moves from negative to positive. |
|
|
Term
|
Definition
-double stranded break will generate linear DNA from either supercoiled or open circular forms
-due to restriction enzyme digestion (where the restriction enzyme cuts the target DNA only once).
-If there are two sites in the target molecule, linear DNA is generated, but it is in the form of two (linear) fragments. |
|
|
Term
PDF 6: Plasmid DNA purification
Suspension in solution 1 |
|
Definition
-Harvest cells and resuspended in Solution containing a buffer (Tris) to stabilize the pH, EDTA and glucose.
-Tris: Buffer to stabilze pH
-EDTA: Chelates cations preventing degradation of to plasmid DNA by depriving endogenous (or exogenously introduced) nucleases of the
cations they require for activity.
Glucose:stabilize the osmotic environment of the cells and prevent premature lysis |
|
|
Term
PDF 6: Plasmid DNA purification
Suspension in solution 2 |
|
Definition
Add solution 2: strongly basic solution (0.2 N NaOH), contains SDS.
-The effect of these two reagents is to lyse the cell and denature nucleic acids and proteins.
-NaOH:denatures macromolecules and disrupts hydrogen bonding between the DNA strands
-SDS: a detergent, which also denatures
proteins and solubilizes lipids |
|
|
Term
PDF 6: Plasmid DNA purification
Suspension in solution 3 |
|
Definition
-A concentrated potassium acetate salt solution at low pH. Two‐fold effect:
-Low pH neutralizes the basic pH in Solution 2
-potassium salt forms a complex with SDS and the molecules to which it is bound. This results in a precipitate containing most of the membranes and chromosomal DNA of the cell. |
|
|
Term
| Plasmid DNA after solution 1, 2, 3 |
|
Definition
| The plasmid DNA, in part due to the fact that it is supercoiled, can rapidly renature and it remains soluble. |
|
|
Term
| How to Remove Percipitate: |
|
Definition
| Centrifugation removes most of the precipitate, and extraction with phenol/chloroform rids the supernatant of soluble proteins. |
|
|
Term
PDF 6: Plasmid DNA purification
ethanol |
|
Definition
| -dehydrates the plasmid DNA, and it can be collected as a precipitate, washed with 70% ethanol to remove excess salt, and resuspended in an aqueous buffer for further analysis. |
|
|
Term
PDF 6: Restriction Enzyme Digestion & Agarose Gel electrophoresis
-type II restriction enzymes |
|
Definition
recognize short, palindromic sequences and make two single stranded cuts at the target site. These may generate 5’ overhangs, 3’ overhangs or blunt ends
see PDF 6 page 10 |
|
|
Term
| PDF 6: What are restriction Enzymes used for? |
|
Definition
-DNA gel blots (Southern blots) to
reproducibly fragment large chromosomal DNA molecules
-important in molecular cloning schemes to prepare both the vector and the target DNA molecules for ligation
-after the subcloning of a fragment from a larger DNA molecule, restriction enzymes are used to determine if the proper fragment has been cloned.
-In some cases, large DNA fragments are subjected to restriction enzyme mapping to determine the linear order of restriction sites. |
|
|
Term
PDF 6: What are restriction Enzymes used for?
-restriction enzyme mapping |
|
Definition
| useful in determining the orientation of the cloned fragment in the vector and the information can be used to decide on the subcloning strategy (i.e., which enzymes might be useful for generating small pieces for subcloning). |
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
transacting factors
DNA binding proteins. |
|
Definition
-Transcription in prokaryotes and eukaryotes involves trans‐acting factors (e.g. RNA polymerase) moving to and interacting with specific DNA sequences (cisacting sequences) to influence gene expression.
-transcription factors often act in combination to control the expression of the gene
-wide variety of sequence‐specific DNA binding proteins coordinate transcription of specific genes or sets of genes to alter the metabolism of the cell or organism. |
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
promoter |
|
Definition
| Region of control sequences that lie ‘upstream’ from the gene. |
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
repressors |
|
Definition
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
inducers |
|
Definition
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
What happens when the environment of the cell changes? |
|
Definition
Responses to the presence (or absence) of hormones, growth factors, and other
metabolites as well as changes in physical parameters such as temperature can quite dramatically alter signal transduction to condition an appropriate response. |
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
operon |
|
Definition
| a cluster of structural genes regulated by a single promoter |
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
What does the Lac operon do? |
|
Definition
| The lac operon coordinates the catabolism of lactose into the simpler sugars galactose and glucose (the preferred carbon source). |
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
What genes does the Lac Operon encode? |
|
Definition
-3 structural genes, genetically referred to as lac Z, lac Y and lac A.
-lac I promoter
-repressor gene
-Lac promoter
-Operator |
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
Lac I |
|
Definition
-lac I gene encodes the lac repressor
-constitutively expressed
-When lactose absent (operon off):
repressor protein binds to the operator sequence and blocks RNA polymerase (no transcription of the genes)
-When lactose present:
lactose binds to repressor protein to prevent it from binding to the operator. |
|
|
Term
PDF 7: Gene regulation in bacteria
-the lac operon
What does cAMP/glucose do? |
|
Definition
-lac operon under positive control from by glucose/cAMP.
-if glucose present, then not energetically efficient to transcribe Lac operon.
Lac operon only on when lactose is present and glucose in sufficiently low concentrations. |
|
|
Term
|
Definition
recognizes its lactose, but also structurally related molecules.
chromogenic substrates: when cleaved they generate a product that is colored and precipitates.
-X‐GAL
-ONPG |
|
|
Term
PDF 7: β‐galactosidase
-X gal |
|
Definition
-5‐bromo‐4‐chloro‐3‐indolyl‐b‐galactopyranoside
-blue indolyl compound as a product when cleaved by β‐galactosidase
-bacteria harboring it will grow and
give rise to blue colonies if X‐GAL is added to the plates |
|
|
Term
PDF 7: β‐galactosidase
-Cloning using X-Gal |
|
Definition
-Plasmid vectors have a portion of the lacZ gene that is interrupted by the multiple cloning site. This interrupts reading from of the Lac Z gene.
If there is no insert in the vector, bacteria harboring it will grow and
give rise to blue colonies if X‐GAL is added to the plates.
bacteria harboring a vector that has an insert give rise to white colonies on X‐GAL containing media. |
|
|
Term
PDF 7: β‐galactosidase
-ONPG |
|
Definition
o‐nitrophenyl‐b‐D‐galactoside
-generates a yellow product when cleaved by
β‐galactosidase.
-used to measure the activity of the
operon. |
|
|
Term
| PDF 8: What are biochemistry techniques used for? |
|
Definition
-purify and characterize various types of small molecules and macromolecules, and to put the properties of these molecules into a biological context.
-how the structure and chemical properties of a molecule determine its structure and function in a living cell |
|
|
Term
|
Definition
| proteins, nucleic acids, sugars, fats and other cellular components |
|
|
Term
| Why use a beaker/flask for mixing solutions? |
|
Definition
-large opening for adding materials to beaker
-may need a funnel to add components to flask. |
|
|
Term
| Advantage in using flask? |
|
Definition
| Can put stopper or fill it to neck to limit evaporation. |
|
|
Term
| How accurate are flasks or beakers? |
|
Definition
| Calibration marks on a typical beaker of flask are accurate to +/-5% |
|
|
Term
| How do you measure a volume accurately? |
|
Definition
-Use graduated cylinder
-use only for measuring or determing liquid volume, not mixing. |
|
|
Term
| How to make an aqueous solution? |
|
Definition
-Start with volume of DIW H2O less than final intended volume
-dissolve one component at a time (perhaps use a magnetic stirrer).
-DO NOT USE HEAT UNLESS SPECIFIED
-When components are dissolved, pour into grad. cylinder.
-add DIWH2O to final volume.
-Pour back into first container to finish mixing. |
|
|
Term
|
Definition
-A capacity close to intended volume of solution.
eg: 100ml sol use 200ml beaker
-for precise volume measurements, use vol. flask (usually not required in bio) |
|
|
Term
| What about solutions that need to be pH'd? |
|
Definition
| -pH prior to final adjustment of volume (adjust to 70% final volume, remaining 30% for pH adjustment) |
|
|
Term
| Why do things need to be pH'd? |
|
Definition
| -biochemical processes occurring in cells and tissues depend on a strict regulation of hydrogen ion concentration |
|
|
Term
|
Definition
biological pH is maintained at a constant value by natural buffers
-in vitro must mimic cell's natural environment
-pH can affect protein structure |
|
|
Term
| What are Biological Buffers, and how are they used? |
|
Definition
buffer ions used to maintain solutions at a constant pH
-are weak acid/base (DO NOT completely dissociate in solution by exists as equilibrian mixtures. |
|
|
Term
|
Definition
| When cells grow, waste products form, so pH can change. Must limit such changes. |
|
|
Term
| What are the most effective buffer systems? |
|
Definition
| -contain equal concentrations of acid (HA) and the conjugate base (A-) |
|
|
Term
| Using Henderson-Hasselbach equation: |
|
Definition
When [A-]=[HA], then pH equals pKa.
pH = pKa + log ([A-]/[HA]) |
|
|
Term
|
Definition
-pKa of a weak acid/base system is the center of the buffering region
-effective buffer range is +/- 1 |
|
|
Term
|
Definition
-must standardize pH meter with buffer solutions of known pH (pH 4, pH7, pH10) before measuring pH of unknown solution
-pH is dependent on temperature, must use temperature correction.
- When concentrated stocks are diluted, must recheck pH as concentration effects pH
-Never let pH meter go dry (put in KCl or pH standard when not in use) |
|
|
Term
| What happens when you overshoot the pH? |
|
Definition
|
|
Term
What happens when you overshoot the pH?
-why? |
|
Definition
| It is not correct to compensate by adding extra acid or base, this will change the solution's composition and cause inconsistency. |
|
|
Term
| What if I overshoot the volume? |
|
Definition
| Do it again to avoid changing the concentration of solutes and to avoid inconsistency. |
|
|
Term
|
Definition
-Select buffer that has a pKa value close to the middle of the range required.
-Adjust pH (pKa) at desired temperature: the pKa of the buffer changes slightly with temperature.
-prepare buffer at working temperature: if prepare stock solutions, make dilutions just prior to use
MAKE BUFFERS AT WORKING CONDITIONS IF POSSIBLE |
|
|
Term
| Why are dilutions important? |
|
Definition
-Need for standard mixture to conduct and assay.
-Need serial dilutions to find the right antibody titer or enzyme activity level.
-Make working solutions from stock solutions (save time)
-Dilute a suspension to desired concentration to clulture cells or to use them in experiments |
|
|
Term
|
Definition
Stored concentrate
-can be used to prepare different working solutions that share the same components by in different proportions
-better for making very dilute buffer solutions (we can pipet a small liquid volume better than weigh a dry chemical)
-can be use as a component of more complex solution. |
|
|
Term
|
Definition
| ration stock concentration to working concentration |
|
|
Term
|
Definition
Can be sterilized, stuffed with cotton for aseptic technique
-To Deliver (DELIVER entire only the amount needed)
-Blow out (entire volume released) |
|
|
Term
|
Definition
BE ABLE TO SPECIFICALLY ASSAY THE THING YOU ARE LOOKING AT
-a physical property that can be observed and measured
-sensitive to change in concentration
-sensitive to specific substance
-changes are predictable and quantifiable
-capable of calibration |
|
|
Term
Assay
eg: How to know the concentration of substance such as DNA, protein, functional enzyme, or other molecules in a volume of solution? |
|
Definition
NEED A DIRECT OR INDIRECT METHOD, BECAUSE CAN'T COUNT MOLECULES.
-use a physical property that changes predictably with concentration of the substance being assayed:
needs to be seen and measured, such as colour, radioactive emission or sound.
must be quantifiable
Must be able to calibrate a detection method. |
|
|
Term
| What range to biological chemical absorb at? |
|
Definition
UV-Visable light range due to resonance of bonds (direct method)
-need relationship between given compound and its absorption characteristics to detect and measure that compound in solution.
indirect (uses colour) |
|
|
Term
| What method to use for protein? for DNA/RNA? |
|
Definition
Protein: indirect
DNA/RNA: direct |
|
|
Term
|
Definition
States that for each unit length of light path, transmitted light is reduced by constant proportion (20%)
As transmittance (T) goes down, absorbance (A) goes up.
A= log(1/T)
A= -log (T)
Both T and A are functions of L (path length) |
|
|
Term
|
Definition
Usually 1cm
-each time you pass thru, lose 20% of light transmitted
-If within accurate range, can just read the absorbance. |
|
|
Term
|
Definition
A:absorbance
T:transmittance
K:CONSTANT related to characteristics (colour, how it is able to absorb transmitted light) of solution
l: path length, usually 1 cm. |
|
|
Term
|
Definition
| ABSORBANCE IS DIRECTLY PROPORTIONAL TO SOLUTE CONCENTRATION AND LIGHT PATH LENGTH. |
|
|
Term
|
Definition
K= ec
K: constant of characteristics of substance
e:extinction coefficient (related to electronic environment or light absorbing unit (chromophore); pH, ionic strength, solvent. Define for a particular solvent at a particular wavelength. Usually INDEPENDENT of concentration.
c: concentration |
|
|
Term
Beer-Lambert Law:
What does it all mean? |
|
Definition
A = Kl =ecl
As e goes up, A goes up. As c goes up, A goes up.
If you know any 3, can solve for the fourth. |
|
|
Term
| Parts of a spectrophotometer: |
|
Definition
Light source (IsubO)
Refracting Prism (separates light into different wavelengths)
Slit (selects wavelength of light.
Chamber (holds substance being measured. Turns IsubO into I)
Photoelectric tube (Detects I and amplifies signal.
Galvanometer (signal converted to read out) |
|
|
Term
| Reliable range of instrumentation |
|
Definition
Needs to be within 0.1-1.0 A
2.3%-90% absorbance
Anything about 1.0 will be out of linear range. |
|
|
Term
Direct spectrophometry:
Pure nucleic acids |
|
Definition
CANNOT DIFFERENTIATE BETWEEN DNA/RNA
Nucleotide bases absorb at 260nm.
For pure nucleic acid:
-1ODsub260=50micrograms/ml of DNA
-1ODsub260=40micrograms/ml of RNA
-1ODsub260= 33micrograms/ml of ssDNA |
|
|
Term
| Why is it difficult to relate absorbance to concentration for TRP/TYR/PHE(aromatic amino acids) residues? |
|
Definition
-Absorb at 280nm
-have different extinction coefficients: difficult to relate to abs to conc.
--approx 0.4-1.5mg/ml of protein per 1OBsub280 (rough rule) |
|
|
