Term
Lab 1: Properties of Macromolecules
Objective: |
|
Definition
| Measure the chemical composition of several urine samples to determine the health state of the individual and practice dilution concentrations. |
|
|
Term
Lab 2: Protein Spectrophotometry
Objective: |
|
Definition
| Practice using spectrometerto measure the concentration of protein in calf serum and understand concentration calculations (Beer-Lambert), using the standard curve, dilution calculations. |
|
|
Term
Lab 3: Restriction Enzyme Digestion
Objective: |
|
Definition
| Practice restriction enzyme mapping and determining whether a mystery plasmid is pBLU or pGLO based on gel electrophoresis results. |
|
|
Term
Lab 4: Bacterial Transformation I
Objective: |
|
Definition
| To practice transformation and insert pGLO bacteria to E. Coli. |
|
|
Term
Lab 5: Bacterial Transformation II and PCR I
Objective: |
|
Definition
Bacterial Transformation II: Determine the growth of colonies on various LB plates.
PCR I: Compare pMCT118 locus of the mystery DNA and my DNA using PCR reaction |
|
|
Term
|
Definition
| Use agarose gel to separate PCR amplified pMCT118 strands and compare to strands left by the anonymous chocolate gifter. |
|
|
Term
| What is the Beer-Lambert law? |
|
Definition
For colored substances in solution, the absorbance is proportional to concentration so A=ECL
A = absorbance rate (slope)
E = molar extinction coefficient, absorbance of a 1M (moles/liter) solution of the substance measured through a 1 cm light path, the constant for each substance at a specific wavelength
C = molecular concentration in moles/liter (M)
L=length of the path light must travel through sample (width of curvette) |
|
|
Term
| What are restriction enzymes? |
|
Definition
| Bacterial enzymes that act as defense mechanisms in the organism by cleaving double stranded DNA internally at specific nucleotide sequences. |
|
|
Term
| How do restriction enzymes defend the cell? |
|
Definition
| By cleaving foreign DNA, like viral DNA, it cuts it into shorter pieces thereby interrupting the foreign genes. |
|
|
Term
| Short Tandem Repeat (STR) |
|
Definition
A DNA sequence (<50) of tandemly repeated short (2-6bp) sequence motifs like GATA. They are used for autosomal profiling, paternity testing and human victim identification. Useful because more DNA found on site will be fragmented and degraded
Cannot distinguish male and female |
|
|
Term
| Single Nucleotide Polymorphisms (SNPs) |
|
Definition
| DNA sequence variation concerning a single base pair in the genome. Uses 50 nucleotides or less to complete a successful analysis though SNPs are less informative when analyzing mixed DNA from multiple individuals. |
|
|
Term
| What makes the pMCT118 locus a good fingerprint marker? |
|
Definition
| It is polymorphic in the general population |
|
|
Term
| For DNA fingerprinting today you will isolate DNA from your own check cells. After collecting the cells you will use heat to... |
|
Definition
| Lyse cells releasing DNA into the solution |
|
|
Term
| If a fluorescent molecule emits yellow light, what color light might have been used to induce fluorescence? |
|
Definition
| Blue, anything with a higher energy (shorter wavelength) than yellow |
|
|
Term
| In the transformation experiment, the purpose of plating cells from the "P4" and "P6" tube on an LB/amp plate is... |
|
Definition
| To determine the concentration of cells that were transformed. |
|
|
Term
Dilution concentration practice problem 1
You take 100µl from "P" tube and dilute in 900µl of LB buffer "A" tube. Then you take 100µl from "A" and dilute it in 900µl of LB in "B" tube. What is your dilution factor from "P" to "B"? |
|
Definition
Dilution factor 10^-2
100µl (P) + 900 µl (A) = 1000 µl total
100µl (A) + 900 µl (B) = 1000 µl total
DF = (P/A) * (A/B) = 1/100 = 10^-2 |
|
|
Term
| What is the purpose of the E. coli origin of replication on the pGLO plasmid? |
|
Definition
|
|
Term
| What is the purpose of the arabinose promoter on the pGLO plasmid? |
|
Definition
| It allows the GFP gene to be expressed when in the presence of arabinose, making arabinose a regulated promoter. |
|
|
Term
| What is the purpose of the ampicillin-resistance gene on the pGLO plasmid? |
|
Definition
| It makes the E.coli resistant to ampicillin, which E.coli normally is not. If E.coli can survive in ampicillin it signifies the bacteria has successfully transformed and taken up the pGLO plasmid. |
|
|
Term
| In the pGLO plasmid, the ampicillin resistance gene is controlled by a constitutive promoter. Why is it important that it is not controlled by a regulated promoter? |
|
Definition
| Because it is an ongoing necessity for the bacteria to always be kept 'on guard" and resistant to ampicillin. |
|
|
Term
| What steps does GFP take to make the GFP protein? |
|
Definition
1. RNA polymerase is recruited by the GFP promoter.
2. RNA polymerase transcribes DNA into mRNA in the nucleus.
3. mRNA is transported out of the nucleus into the cytosol.
4. mRNA is translated by cytosolic ribosomes into a protein.
5. GFP protein is exposed to UV light, which causes it to glow green. |
|
|
Term
| With the exception of glycine, all of the R groups from the amino acids in the chromophore region of both GFP and eGFP are _____ and they share the functional group ______. |
|
Definition
|
|
Term
| What amino acids are key in forming the GFP protein and how does the structure fit the function? |
|
Definition
| Serine, Tyrosine, and Glycine are used in building the GFP protein. These amino acids interact within the beta barrel in specific ways that allow them to rearrange to form the chromophore. |
|
|
Term
| What is the visible light spectrum? |
|
Definition
Red: 680nm-610nm
Orange: 610nm-590nm
Yellow: 590nm-560nm
Green: 560nm-520nm
Blue: 520nm-500nm
Violet: 500nm-400nm |
|
|
Term
| What wavelength best corresponds to red light? |
|
Definition
|
|
Term
How do you calculate standard curve?
How do you calculate E using the standard curve? |
|
Definition
standard curve = E ≈ A/C
E is also the slope
E = ∆A/∆C |
|
|
Term
| What does the standard curve measure the relationship between? |
|
Definition
| It helps us determine the concentration of a substance and the absorbance of that substance at a specific wavelength. |
|
|
Term
| True or False: If a substance appears blue it absorbs mainly blue wavelengths of light. |
|
Definition
|
|
Term
| If a solution is too weak or too strong how do you find A? |
|
Definition
| Use a standard curve by drawing a line of best fit after measuring and plotting the absorbance |
|
|
Term
| How do bacteria protect their own DNA from digestion from their own restriction enzymes? |
|
Definition
| Bacteria chemically modify certain bases in their own DNA that prevent restriction enzymes from cleaving. |
|
|
Term
| If you perform agarose gel electrophoresis with a mixture of DNA fragments the smallest ones will... |
|
Definition
| Migrate fastest and be found at the bottom of the gel. |
|
|
Term
| True or False: both SYBR green and orange G bind to DNA |
|
Definition
|
|
Term
| What's the purpose of using a DNA marker when performing agarose gel electrophoresis? |
|
Definition
| To give a standard comparison of where known base pair segments lie. |
|
|
Term
| If plasmid pBAR is 5000 bp and it is cleaved by EcoRI at target sites 500 and 2500, how many fragments and what size fragments are produced? |
|
Definition
| Two fragments: 2000 bp and 3000 bp |
|
|
Term
| If bacteria transformed with pGLO plasmid were plated on LB media with ampicillin but not arabinose which genes would be expressed? |
|
Definition
| The ampicillin resistance gene. |
|
|
Term
|
Definition
| When bacteria take in material from their environment and incorporate it into its own DNA |
|
|
Term
| What is the purpose of using calcium chloride in our transformation experiment? |
|
Definition
| CaCl2 is used so that Ca+2 (positively charged) will act as a bridge to allow negatively charged DNA to enter the cell through pores in the negatively charged phospholipid layer. |
|
|
Term
| Summarize "Three Papers, Three Lessons" |
|
Definition
1. It is helpful and encouraged to work with colleagues and look at their research.
2. Unpublished research will not be credited.
3. Write precise, short, and clear lab reports. |
|
|
Term
|
Definition
| The study of the chemical properties of biological molecules. |
|
|
Term
| What percentage of the cell is water and what percent is dry matter? |
|
Definition
|
|
Term
| What are the four classes of organic compounds in monomer and polymer form? |
|
Definition
proteins: amino acids and polypeptides
carbohydrates: monosaccharides and polysaccharides
lipids: fatty acid and fats
nucleic acid: nucleotide and nucleic acids |
|
|
Term
| What is the excretory system? |
|
Definition
| The systematic disposal of metabolic wastes and the control of body fluid composition. It is comprised of kidneys, ureters, urinary bladder, and urethra. |
|
|
Term
| What are the main functions of the kidney and how does it do this? |
|
Definition
1. Maintain ion concentration and water volume
2. remove waste
All through filtration, selective reabsorption, and excretion. |
|
|
Term
| What are the parts of the kidney? |
|
Definition
| Nephron: Bowman's capsule, glomerulus, proximal convoluted tubule, Henle's loop, distal convoluted tubule |
|
|
Term
| What and where are the processes within the nephron? |
|
Definition
1. filtration (blood enters the glomerulus)
1.5. Blood flows to the Bowman's capsule. Blood cells and proteins do not enter
2. reabsorption (water, glucose, salts, and amino acids are taken into the blood in the proximal convoluted tubule)
3. secretion (excess ions travel through distal tubule)
4. release (collecting duct --> end of nephron) |
|
|
Term
| What are two advantages to the filtration/reabsorption system? |
|
Definition
1. high capacity to remove unwanted substances on a moment to moment basis
2. ability to eliminate a broad range of substances in one go |
|
|
Term
| What substances should not be present in urine? |
|
Definition
|
|
Term
| How do we measure organic compounds that are generally colorless? |
|
Definition
Biuret reagents reacts with compounds and produce a color depending on the concentration of the protein.
Ex. proteins turn purple
Ex2. amino acids turn pink or purple depending if they are free or linked to proteins |
|
|
Term
| What is the equation for dilution calculations? |
|
Definition
(c1) (v1) = (c2) (v2)
c1 = starting concentration
v1 = starting volume
c2 = final concentration
v2 = final volume
c1 and c2 must have the same units |
|
|
Term
Dilution concentration practice problem 2
You are provided with hemoglobin at a concentration of 10 mg/ml. If you mix 5 l of that solution with 10 ml of saline, what would the concentration of hemoglobin be in the diluted solution? |
|
Definition
c1 = 10 mg/ml; v1 = 5 ml
c2 = X; v2 = c1+v1 = 5 ml+10 ml = 15 ml
(c1) (v1) = (c2) (v2) --> (10mg/mL)(5mL) = X(15mL)
X = c2 = 3.3 mg/ml |
|
|
Term
| What is the dilution factor? |
|
Definition
|
|
Term
|
Definition
| Substances that absorb visible light. |
|
|
Term
Which is qualitative and which is quantitative?
1. Is there protein in the urine?
2. How much protein is in the urine? |
|
Definition
1. qualitative
2. quantitative |
|
|
Term
| What are the parts of the spectrophotometer? |
|
Definition
spectrometer, photometer (photoelectric tube, galvanometer)
SPPG
Simple People Play Gameboy |
|
|
Term
| What is the spectrometer? |
|
Definition
Provides discrete wavelengths of light at known intensities (incident).
Uses white light source and a monochromator (prism) |
|
|
Term
|
Definition
| photoelectric tube (reads wavelength of light emitted by spectrometer) and galvanometer (measures quantity of intensity of transmitted light) |
|
|
Term
| How does a spectrophotometer work? |
|
Definition
1. Spectrometer directs incident light (specific wavelength) into the solution.
2. Transmitted light (light that passes through the solution) is sensed and quantified by photometer |
|
|
Term
| What is the absorption spectrum? |
|
Definition
A distinct set of absorbable wavelengths
*Produces a pattern of peaks and valleys of light absorption
*What is absorbed is not seen. What is reflected is seen. |
|
|
Term
| What are two ways we use restriction enzymes? |
|
Definition
1. modify and manipulate DNA molecules to construct recombinant DNA
2. create a restriction map (distribution of target sites for various restriction endonucleases) which is important in analyzing cloned DNA |
|
|
Term
|
Definition
| Small circular pieces of DNA that contain genes (regions of DNA that transcribed into mRNA and then translated into protein) |
|
|
Term
|
Definition
| Regions of DNA that control whether or not a gene is transcribed. They work by recruiting RNA polymerase to a specific area of DNA so that the gene of interest can be transcribed into mRNA |
|
|
Term
| What is gel electrophoresis? |
|
Definition
| A method of separating DNA fragments based on size by running an electric current through agarose gel. |
|
|
Term
| What is an alternative to gel electrophoresis? |
|
Definition
|
|
Term
| Why does gel electrophoresis require a current? |
|
Definition
The current runs negative --> positive.
DNA is negatively charged (in basic pH environments) so they will migrate towards the positive end.
The closer to the positive pole, the faster they migrate |
|
|
Term
| What determines the relative rate of migration through the gel matrix? |
|
Definition
| 1. Size: smaller molecules have more ease working through the gel matrix 2. Proximity to the positive pole |
|
|
Term
|
Definition
1. SYBR green (binds to DNA and fluoresces)
2. orange G (makes the progression visible)
3. glycerol (allows sample to sink to bottom of the well) |
|
|
Term
| What is the importance of a marker/ladder? |
|
Definition
| These known sizes of DNA are important to compare your data to |
|
|
Term
| What does it mean if bacteria is competent for transformation? |
|
Definition
| It is naturally able to take up DNA from the environment. |
|
|
Term
| Why is natural transformation important? |
|
Definition
| It allows the transfer of genes between bacteria creating lateral gene flow. In biology it allows for chemical and biological analysis of those molecules. |
|
|
Term
| What is an expression vector? |
|
Definition
Plasmids that are designed to allow for expression of specific genes in an organism.
Ex. pGLO allows for the expression of GFP in E. coli |
|
|
Term
| Why is pGLO considered a recombinant DNA molecule? |
|
Definition
| Because it contains DNA sequences from two different organisms, E. coli and GFP |
|
|
Term
| What is a selection gene and why is it important it is in expression vectors? |
|
Definition
The selection gene allows bacterium to survive and multiple in a given environment.
Ex. For pGLO plasmid the selection gene is amp(r) that makes a protein that confers resistance to the antibiotic ampicillin. E. coli typically cannot survive in ampicillin.
If E. coli express the resistance gene (amp(r)) then we know that the expression vector has also been taken up. |
|
|
Term
| When are genes controlled by constitutive promoters expressed in the cell? |
|
Definition
Always because these genes are needed on an ongoing basis.
Ex. amp(r) in E. coli |
|
|
Term
| When are genes controlled by regulated promoters expressed in the cell? |
|
Definition
Only under certain conditions because it is a waste to expend energy if it is not necessary.
Ex. Arabinose is only turned on by E. coli when needed (in the presence of abundant arabinose) |
|
|
Term
| Where do promoter sequences need to be placed in order to control gene expression? |
|
Definition
| Immediately upstream of a gene. |
|
|
Term
| Can you use a regular promoter from GFP to express GFP in a bacterial cell? |
|
Definition
| No, you must use a bacterial promoter. Regular promoter for GFP comes from jellyfish so it would not be recognized by bacterial RNA polymerase. |
|
|
Term
| What are adhesion zones in bacterial cells? |
|
Definition
| Tiny pores in the plasma membranes |
|
|
Term
| What is GFP controlled by in bacterial cells? |
|
Definition
| A regulated bacterial promoter called P(ara). It is turned on in the presence of arabinose and activates the genes downstream (GFP) |
|
|
Term
What will grow on the various types of media and which will express GFP?
1. LB agar
2. LB agar+amp
3. LB agar+amp+ara |
|
Definition
1. no growth (no amp)
2. growth but no expression of GFP (no ara)
3. growth and expression of GFP |
|
|
Term
| What is polymerase chain reaction (PCR)? |
|
Definition
| A quick and efficient technique that allows for the isolation of any specific DNA fragments (as long as you know the DNA sequence). It can be performed in vitro. |
|
|
Term
| What is the difference between in vivo and in vitro? |
|
Definition
in vivo (in a living cell): hydrogen bonds holding the two DNA strands are broken by DNA polymerase and other proteins. Primase generates primer strands
in vitro (in PCR reaction): hydrogen bonds holding DNA strands together are broken apart using heat. Primer strands are synthesized and only contain the target gene. |
|
|
Term
| How many primers are used in PCR? |
|
Definition
two, one for each DNA strand.
primase: runs 5'-->3'
reverse primase: runs 3'-->5' |
|
|
Term
| What are the steps to the PCR cycle? |
|
Definition
1. four main components are mixed together
2. heating: mixture is heated to 92-96˚C (break hydrogen bonds)
3. annealing: mixture cools and two stranded DNA molecules reform
4. replication: Taq polymerase extends each primer in the 5'-->3' direction using nucleotides in the mixture
repeat 30-40 times |
|
|
Term
| What are the four main components in the PCR mixture? |
|
Definition
1. many copies of the two primers
2. multiple copies of the target DNA
3. mixture of four DNA nucleotides (dATP, dCTP, dGTP, dTTP)
4. Taq polymerase (special heat resistant DNA polymerase) |
|
|
Term
|
Definition
Two or more alleles of a locus that exist in the human genome in a given population.
Ex. pMCT118 |
|
|
Term
| Define single nucleotide polymorphisms (SNPs) |
|
Definition
| Polymorphisms with just a single base pair difference |
|
|
Term
| Why is pMCT118 referred to as VNTR? |
|
Definition
VNTR: variable number of tandem repeats
The number of times pMCT118 is repeated (14-40) on the genome various depending on the individual because individuals are heterozygous at this locus. |
|
|
Term
| What is a short tandem repeat (STR)? |
|
Definition
| A polymorphic region of the genome in which very small repeats (3-4 base pairs) are repeated |
|
|
Term
| What does it mean if an individual is heterozygous at the pMCT118 locus? What will our results show? |
|
Definition
It means she receives a different number of repeats of pMCT118 from her mother and father.
Ex. 14 copies from mom, 34 copies from dad
yields two different size PCR products, yields two different bands in agarose gel |
|
|
Term
| What is the significance of polymorphic loci? |
|
Definition
| It allows for DNA fingerprinting, diagnosis for inherited disorders, paternity testing, etc. |
|
|
