BIOL LAB FINAL

Describe the basic process of an ELISA. What binds to what? When you get a positive or negative result from an ELISA, what are you actually seeing?

+ Antigen → primary antibody → secondary antibody → conjugated enzyme --> substrate

It also involves an antibody or antigen (immunologic molecules) that may form an antigen-antibody reaction to provide a positive result or, if they do not react, a negative result.

Positive control show color blue = substrate binds to conjugated enzyme of secondary antibody resulting existence of antigen in the well.

Negative control shows nothing is bound in the reaction showing the clear color indicating no antigen there.

Which of the following errors would result in positive results for all samples in an Elisa assay (including negative controls). How to perform ELISA?

failure to perform the wash step after incubation with secondary antibody. --> Add sample to wells. Allow proteins and any antigens (if present) to bind to wells (adsorb), by hydrophobic interaction. Add primary antibodies to bind to antigen. add secondary antibody with enzyme onit. Allow secondary antibody to bind to primary antibodies. Add substrate. Substrate will bind to enzyme, and detect presence of secondary antibody. If secondary antibody is present we can say the antigen is present. + Wash with buffer twice after step 1 & 2. + wash with buffer twice after step 3. always tap out liquid before adding more.

Which errors result in negative results for all samples in an Elisa (including positive controls)?

failure to add antigen, f to add functional primary antibody, secondary antibody & functional substrate

Reusing a pipet tip when transferring controls & samples from tubes to wells

is more likely to cause false positives than false negatives

What are negative and positive controls? And what are their functions?

Positive control = to make sure that all reagents we're adding bind together and gives us positive control

- Also looking at color change to determine results

Negative control = to check for no contamination, antigen or antibody is missing

Given wells containing tested samples, be able to classify them as positive or negative results.

Positive test: presence of serum antibodies against a specific pathogen indicates that the patient has encountered a disease + Immune system produces antibodies goes against antigen (foreign HIV). If antibodies from patient blood binds to antigen meaning patient has disease (Positive test would show color change and antigen existed)

Be able to recognize erroneous results and determine potential source(s) of error.

Suppose assay was blue in first well, but clear in second well of negative control samples. Possible errors?

Cause false + result: immunoassays for antibodies to human immunodeficiency virus (HIV), and can result from recent vaccinations

Cause false - result: negatives can result from immunosuppression (when the immune system suppress the transplant liver/AISD in the body)

--> reused pipetting causing contamination & false positive result

Understand the purpose of a PCR reaction

+ PCR is valuable to researchers because it allows them to multiply unique regions of DNA so they can be detected in large genomes.

+ Thanks to this procedure, one can make billions of copies of a single DNA molecule even though it is initially present in a mixture containing many different DNA molecules.

+ The purpose of a PCR reaction is to amplify small amounts of a DNA sequence of interest so it can be analyzed separately.

Understand the mechanism of a PCR reaction

a) List essential components of a PCR reaction

+ Target DNA

+ A pair (one for each strand) of primers specific for the target DNA

+ Free deoxynucleotide

+ Heat stable DNA polymerase (Taq)

+ Buffer (containing cofactor Mg+)

Understand the mechanism of a PCR reaction

What kind of DNA can be used as template? Does it have to be pure?

+ DNA that is specific to the primers that are being used can be used.

+ Both crude and pure DNA can be used b/c primers are specific and will attach to its designated DNA.

- Must be complementary to the 3' end of the gene to be amplified (specific to target DNA)

Understand the mechanism of a PCR reaction

The basic properties of PCR primers

2 types of primers are needed (one for each of the strands)

+ They are around 20 nucleotides long

+ Designed to bind to the region that needs to be amplified

+ Must be complementary to the 3' end of the gene to be amplified (because transcription occurs from 3' to 5') (new nucleotides are added from 5' 3')

+ Quantity: in large excess

Why should there be primers in the reactions?

Because DNA polymerases cannot copy a chain of DNA without a short sequence of nucleotides to "prime" or start the process

The primers are needed to match the desired DNA segment to be copied, and they allow replication to occur.

What is special about the polymerizing enzyme used in the reaction?

The polymerase that is used in PCR is called Taq polymerase

+ It is special because it can survive high temperatures

+ This allows it to survive through many cycles of PCR which are carried out at high temperatures

What are the three basic steps in each cycle?

The temperature used and the purpose of each step

1. Denaturation at 94°C - separation of a DNA strand into 2 single strands

2. Annealing at 58°C - the attachment of primers to the ends of the single strands of DNA

3. Primer extension at 72°C - synthesis of new strands; makes copies of templates

Is the amplification linear or exponential?

o Exponential (2^n where n= the number of cycles)

o At the end of each cycle the amount of DNA doubles

o After 35 cycles you will get 2^35 copies of DNA or 34 billion

What determines the size of a PCR product?

+ SIZE OF THE PRIMERS: To find the size of the PCR product you need to find where the right and left primers are set. In this lab the right primer was set on 16,411 and the left primer was set on 15,971 base pair. The difference between the two values determines the size of the PCR product. So 16,411-15,971 = 440 base pairs.

+ The placement of the two primers regarding the gene of interest will determine the size of the product (difference of the location of the two primers). You want to place the primers on both ends of the DNA segment you want to copy.

What are the two functions of the dNTPs?

a) Provides building blocks for new DNA strands to be copies during PCR

b) Function as an energy source for reaction

Why is MgCl 2 included in the reaction?

+ MgCl 2 is included in the reaction because it is a cofactor (Mg 2+ is a cofactor) for Taq polymerase enzyme and acts as a catalyst.

+ It acts like a catalyst to the polymerase and helps speed up the process. It is a cofactor for Tag polymerase. The Mg++ helps stabilize the two strands. Normal concentration of Mg++ is 2.5um; higher concentration yield greater errors and lower increase the specificity of the target that you are trying to amplify.

Understand how you set up your PCR reaction in the lab.

a) Obtain PCR reaction tube (containing a small Ready-to-Go PCR bead)

b) Use a micropipette (w/ fresh tip) to add 20 μl of the appropriate primer/loading buffer mix to PCR reaction tube. Tap tube to dissolve bead (bead contains Taq polymerase)

c) Use red fixed-volume micropipette and fresh tip to add 5.0 μl of human DNA to PCR reaction tube and tap to mix

d) Label cap of tube with sign-in # and section #

e) Place PCR reaction tube in ice bucket next to the PCR machine

Isolation of total DNA from epithelial cells (the function of Chelex and boiling)

Chelex ensures the removal of PCR inhibitors (contaminating metal ions that catalyze the digestion of DNA)

Boiling the cell mixtures helps to break up the cells and denatures proteins to get your desired DNA strands.

→ Serves to lyse (dissolution) the cell to release the DNA and at the same time, inactivate cellular proteins that may interfere with the PCR reaction.

A student performed a PCR reaction and he wants to know if he has obtained the expected PCR product. What does he need to do?

The student needs to analyze his PCR product through gel electrophoresis (DNA ladder is also used in this process). Gel electrophoresis will show the student roughly how many base pairs are in his product when he compares it to the DNA ladder on the gel.

What is the basis of separation of DNA on an agarose gel? How is it different from the protein agarose gel (E11) in terms of the setup or the property of the gel?

+ Agarose gel separation in this lab relies on the size of the DNA fragment; Smaller fragments travel further

+ In experiment 11 the protein agarose gel relies on the charge of the fragment; You also place the comb in the middle of the protein gel instead of the end like you do

for the DNA agarose gel, and you don't use a ladder for protein.

+ Other factors that influence the rate of migration include: the conformation of the DNA, the agarose concentration, the buffer and the applied voltage

What gives DNA the negative charge?

Phosphate groups are negatively charged thus making DNA negatively charged (migrate to anode)

Understand how your PCR product was visualized

a) What affect the migration rate of DNAs in agarose gel electrophoresis?

+ The size of the DNA and the shape of the fragments (circular vs. linear) affect

the migration rate of DNA molecules in agarose gel electrophoresis.

+ Other factors include: agarose concentration, buffer, applied voltage

What chemical "stains" the DNA in our experiment? And how

Ethidium Bromide

+ It binds to the DNA molecules

+ It is fluorescent under UV lamp

What is the purpose of including a marker lane in the gel?

The purpose of including a marker lane in the gel is to give the individual something to which they can compare their PCR products to estimate the size of their fragment measured in base pairs.

How does the size of linear DNA molecules relate to their migration rate?

+ Smaller DNA fragments migrate further/faster

+ The mobility of the linear DNA fragments is inversely proportional to the LOG10 of the molecular weight

What two blue dyes are included in the marker? Why? (page 3 in E5 procedure)

+ Loading buffer usually contains something dense (e.g. glycerol) to allow the sample to fall; into the sample wells.

+ It also contains one or two tracking dyes, which migrate in the gel and allow visual monitoring of how far the electrophoresis has proceeded.

+ The dye fronts migrate toward the positive end of the gel, just like the DNA fragments.

+ The "faster" dye, Bromophenol blue, co-migrates with DNA fragments of approximately 300 bp, while the "slower" dye, Xylene cyanol, co-migrates with DNA fragments approximately 9 kb in size.

→ Bromophenol blue and Xylene cyanol are included in the marker because they allow visual monitoring of how far the electrophoresis has proceeded in the experiment

Define genetic transformation

The ability to introduce a specific gene into an organism and have it express that gene

Compare constitutive vs. inducible gene expression.

+ Constitutive systems are always on, lack regulation. ex: the gene for the repressor protein in prokaryotes which sits at the operator for lac operon.

+ Inducible systems are turned on in the presence of a molecule that needs to be broken down. Enzymes only produced when specific chemical substrates are present.

Ex: lac operon in prokaryotes.

Inducible system

- Arabinose present in growth media

• The gene for GFP will be switched on

• Transformed cells will appear fluorescent green under UV light

- No arabinose present in growth media

• The gene for GFP will remain switched off

• Transformed cells will appear white under UV light

What are some important characteristics of the strain and the plasmid in this experiment?

We used E.Coli bacteria and placed a unique pGLO plasmid that codes for GFP and bla, which provides antibiotic resistance. pGLO can be used to control expression of fluorescent protein in transformed cells. E.Coli was used because it is a single celled organism that produces quickly and will not infect plants or animals and it cannot grow on plates with antibiotic (ampicillin)

Understand how bacterial transformation is set up in this lab.

a) Why do we use LB/amp media to select for transformants?

The bacteria with the pGLO DNA also codes for beta lactamase, which allows for the bacteria to be resistant to ampicillin. The ones that did not transform would be killed.

What was the purpose of the arabinose?

If Arabinose is present, it turns on the beta lactamase gene of the PGLO plasmid which is an antibiotic resistant gene. This phenomenon allows the transformed cells to grow even in the presence of ampicillin.

Which plate---LB/amp or LB/amp/arab--- produced glowing colonies? WHY?

LB/amp/arab produced glowing colonies because the arabinose activated the gene so it could be expressed which means glow.

What are the control plates? What are their purposes?

the LB plate was used to ensure that we had live bacteria & they were not killed off during the process.

+ LB plate with no plasmid (pGLO) demonstrates Positive control with TMTC

+ LB/AMP plate with no plasmid (pGLO) demonstrates Negative control with no growth

Be able to determine the transformation efficiency given the number of transformants, the concentration and volume of plasmid used.

a. Total amount of pGLO DNA=concentration of DNA (ug/uL) * volume of DNA in uL

b. Fraction of DNA used= Volume spread on plate/to sample in microfuge tube

c. DNA spread = fraction of DNA * total DNA used

d. Transformation efficiency = number of cells on plate / DNA spread

Four different treatments of bacterial cells that enhance transformation efficiency

a. Incubation

i. Cells mixed with CaCl2 + plasmid

b. Heat shock

i. Quick temperature changes (destabilizes cell membrane to create thermal imbalance)

c. Recovery

i. Short incubation to begin expressing new genes

d. Cells of log phase

Sample question:

A single colony of bacteria HB101 was mixed with 800 μl of CaCl 2 transformation solution and split into two tubes (+pGLO and -pGLO). 20 μl of plasmid pGLO DNA with concentration of 0.03 μg/μl was added to +pGLO tube. After heat shock, 180 μl LB broth was added to each tube.

150 μl of the cell suspension from (+) pGLO tube was spread onto a LB/Amp plate, and 300 transformants were observed on that plate after incubation.

Transformation efficiency = transformants/μg of plasmid DNA

Total amount of pGLO DNA used = __________

Fraction of DNA used =________________

Micrograms of DNA spread on the plates =____________

Transformation efficiency =________________

0.6 μg, 0.25, 0.15 μg, 2000 transformants/ μg

Know how to prepare a dilute solution from a stock solution using parallel dilution method.

(C1)(V1) = (C2)(V2)

C = concentration

V = Volume

Need to know the initial concentration and final volume

Know what an absorption spectrum is AND how it is generated. Be able to graph the data into a properly labeled curve. (title, label, units of measurements, linear scale).

Used to determine lambda max

X axis is wavelength (in nm)

Y axis is absorbance (no units)

Understand the concept of max of a chemical and how it is determined experimentally.

+ Lambda max is where maximum absorbance occurs for a given substance

+ Use an absorption spectrum to determine it

+ We measure the lambda max so that we can be accurate and sensitive in our experiments when using the spectrophotometer

+ Experimentally this can be determined by taking a series of readings at different wavelengths and finding the wavelength that gives you the highest reading.

The following questions are all related to the standard curve showing the relationship between absorbance and concentration:

What is the purpose of the standard curve?

+ It is measured at the lambda max for all values

+ You only auto zero once (because you are not changing the wavelength)

+ It should be a straight line

+ The equation of the line should be in y=mx+b

+ Where, Absorbance = (constant)(concentration)

+ Y axis is absorbance

+ X axis is concentration (in micrograms)

+ Based on beers law

+ It is used to identify the concentration of an unknown sample using its measured absorbance

+ The curve relates the absorbances to the concentrations of a chemical solution.

Why such a curve is typically measured at the max of a chemical?

The curve is measured at the λmax because that is the point where the absorbance is most sensitive and will be the most accurate. This wavelength should be used to calculate all other absorbance values because it will yield the best results.

Be able to describe the Beer's law and relate it to the standard curve

A = bec

+ e = molar extinction coefficient which constant dependent on both wavelength & substance (Lmol^-1cm^-1)

+ b= pathlength (cm)

+ c = molar concentration (mol/L)

+ A = absorbance (no unit)

Be able to manually draw and properly label a standard curve provided with data

+ Y axis is absorbance (no units)

+ X axis is concentration (in Mm)

"absorbance vs. concentration of X substance at (insert lambda max)"

Be able to use the standard curve equation generated by Excel to calculate the concentration of an unknown solution.

+ The standard equation generated by excel is in the form y = mx

+ Absorbance = (constant)(concentration)

+ Solve for missing variable

Wavelength ranges describes visible light?

400-700 nm

A spectrophotometer measures

absorbance of light

An absorption spectrum KMnO4 will be generated in E9 experiment. What reported on the X- axis?

Wavelength

A standard curve for KMnO4 will be generated in E9 experiment. What on the X- axis?

Concentration

We will discover the lambda max (λmax ) of a chemical solution by generating and analyzing the absorption spectrum.

True

What are the two reasons for measuring the absorbance of the sample at its λ max?

Absorbance is more accurate, the error in determining concentration will be lowest, Better sensitivity to distinguish two similar concentrations of the same chemical compound

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

Seven solutions will be prepared with the mixture of distilled water and stock solution of KMnO4 (1 mM) as listed in Table 1. Fill in the blanks with the correct volumes for the solutions listed below. The final volume in each tube should be 2ml. If a value is less than 1, you must place a zero before the decimal point, ex: 0.7 Tube C (0.1 mM) will be made with [a] mL of 1mM KMnO4 stock and [b] mL of distilled water. Tube F (0.3 mM) will be made with [c] mL of 1mM KMnO4 stock and [d] mL of distilled water.

0.2, 1.8, 0.6, 1.4

What may influence the activity of an enzyme? List at least 4 factors.

Temperature

pH

Amount of enzyme

Substrate concentration

Effects of inhibitors and/or activators

Describe how the amount of enzyme affects the rate of a reaction.

The more abundant the enzyme the quicker the products is formed

Describe how the amount of substrate affects the rate of a reaction.

As long as the substrate concentration increases, there is a direct increase in the rate of the reaction. In effect, the rate of the reaction is limited by the speed with which the substrate is converted into product and thus on the availability of the enzyme.

Understand why reaction rates decline with time and use this information to correctly process the data (by choosing the proper data points to do linear regression)

a. depletion of substrate

b. breakdown of enzyme

c. presence of inhibitors

Understand how to construct and read the following two graphs:

The standard curve of absorbance vs nmole/ml of nitrophenol

The amount of nitrophenol produced in the assay vs time

The standard curve of absorbance was used to estimate the concentration of nitrophenol based on the OD410. the amount of nitrophenol vs time graph is to get the rate of the reaction over time. Usually we use the beginning part of the graph to get an accurate reading of how fast the enzyme works.

Be able to describe the reaction catalyzed by acid phosphatase and describe how the assay was performed, and the functions of KOH in the assay.

a. The acid phosphotase cleaves a phosphate group from the large molecules. Nitorphenyl phosphate is colorless, but when it is converted into Nitrophenol it becomes yellow in alkaline solution by KOH.

i. An enzyme reacts with a substrate forming a substrate complex. The enzyme is specific to certain substrates. Once it has converted the substrate into the product, it moves on to another substrate.

ii. KOH serves two purposes

1. Stops the reaction because enzyme needs acidic conditions, makes it alkaline condition (yellow color)

2. Gives for color formation, turning it yellow

1. Understand the subunits of proteins: amino acids

Provided with the structure of amino acids, be able to classify them into non- polar, polar or charged amino acids

Know which two amino acids are acidic amino acids, which three amino acids are basic amino acids, under what condition?

i. Acidic amino acids are the following under neutral pH

1. aspartic acid

2. glutamic acid

ii. Basic amino acids are the following under neutral pH

1. lysine

2. Arginine

3. Histidine

Any amino acid can be charged under appropriate condition. True or False?

TRUE

Define the four levels of protein structures.

a. Primary = the basic polypeptide chain of amino acids. Linear fashion

b. Secondary = includes helices and beta sheets

c. Tertiary = The alpha helices and beta sheets react with each other making a 3d structure

d. Quaternary = multiple tertiary proteins come together (2 or more)

Understand the concept of pI

the point at which the protein does not migrate in an electric field (isoelectric point).The pH at which net charge of protein = 0

Know the relationship between acidity of amino acid or proteins and pI

If the amino acid is positively charged = acidic (lower pI)

If the amino acid is negatively charged = basic (higher pI)

pH < pI → amino acid is positive charged

pH > pI → amino acid is negative charged

Be able to determine the charge of an amino acid or a protein given its pI and environmental pH ; then predict its migration direction in an agarose gel

+ if the pI is larger than the pH then the amino acid is positively charged. It will move toward the cathode.

+ if the pI is smaller than pH then the amino acid is negatively charged. It will move toward the anode.

Understand the difference between normal hemoglobin and mutated hemoglobin in sickle cell patients; be able to compare the migration rates of the two types of hemoglobin (HbA and HbS) based on the nature of the mutation.

In hemoglobin S (sickle cell anemia) a single glutamic acid residue on the Beta chain is replaced by valine. This causes the HBB sequence to go from HbA to HbS. This causes the net charge to change and the shape of the protein. The Hemoglobin S is deoxygenated. The hemoglobin molecules are negative charge and move toward the anode when the buffer pH is at 9.2. The isoelectric point of normal hemoglobin is 6.9, but hemoglobin S has a distinct two fewer negative charges. This means that it would move less distance to the anode compared to normal.

Understand the separation principle of protein agarose gels (native or non-reducing gel electrophoresis)

What determines the direction of movement of the proteins?

the migration of the protein through an agarose gel is dependent on the net electric charge on the protein, intensity of the electric field, and the pH of the ionic strength of the buffer

What determines the rate of movement of the proteins?

the rate of movement of the proteins is determined by the difference between the isoelectric point and the pH of the buffer.

Does the molecular weight of a protein affect its migration?

No

Does the separation require proteins to be denatured?

No

Does the protein change its shape while it is moving in the gel?

No

Properly load a sample into an agarose gel. Assemble the gel apparatus and connect to the power supply correctly (the wells was set at the end of the gel).

Check lab manual & review

Primers need to be designed

outside

primer design PCR can anneal to DNA of both human epithelial cells and bacteria collected in mouthwash

False, it only recognize human cell

5' CATGGATG 3' is complementary to

5' GCATGAATGCATGCA ... TGCATCCATG3' so it can be used as one of the primers in pCR to amplify this DNA region

True

What is the direction of synthesis of the new DNA strand in each cycle?

from 5' to 3'

What determines the size of the final PCR product?

2 locations of the 2 primers to find the size

What temperature is used to denature the DNA template?

95C

What temperature is used to synthesize the new DNA strand?

72C - 75C

What is unique about the Taq DNA polymerase?

Heat resistant, thermostability

Do you need to have a pure template DNA sample (free of other DNA sequences) to allow specific amplification of a segment of the template DNA?

No because PCR only works for human DNA (they have been programmed specifically)

What would be the consequence if the reaction contained proteases (enzymes that can degrade proteins into amino acids)?

If proteases were present, those enzymes would degrade Taq, thus PCR would fail

What would be the consequence if the reaction contained DNases (enzymes that can degrade DNA into nucleotides)?

If DNases were present, those enzymes would degrade the DNA components, then PCR would fail also

What two components need to be added to the puReTaq Ready-To-Go PCR Beads to set up a PCR reaction?

1) a new pair of primer/ loading buffer mixed 2) DNA template (from yourself)

(True or False) The amplification of the original target DNA is exponential.

True

What are the three main steps in procedures of transformation?

Incubation, heat shock & recovery

What methods can be used to make cells permeable to plasmid DNA?

Heat shock, microprojectile, calcium chloride

Describe the important characteristics of the pGLO plasmid and host cells in this experiment?

+ pGLO plasmid: contains selectable marker (ampicillin resistant gene which codes for beta-lactamase), origin of replication, polylinker area, gene for GFP, and BONUS gene regulation system for GFP (inducible)

+ Host E. coli: single-celled, reproduces quickly, does not infect plants and animals, and not ampicillin resistant

Which is the selectable media for transformants and which media will produce growing colonies? Why?

+ LB provides nutrients to grow while ampicillin kills the non-transformed cells, allowing just transformants to grow

+ LB/Amp/pGLO/ara because amp kills non-transformed cells, pGLO provides GFP when arabinose induces it to express, GFP causes glowing

Host cells carrying the pGLO plasmid can grown on LB plates containing ampicillin because __

Beta-lactamase express in transformed cells

If you mix pGLO DNA with arabinose in a microcentrifuge tube will you observe fluorescence under UV light?

NO WAY!

In order for RNA pol to efficiently transcribe GFP mRNA from the pGLO plasmid you need

araC Protein

Vo is the __

rate of the reaction

Optimum pH =

the point where enzyme is most active

All spectrophotometer readings for this experiment will be taken at 410 nm why?

410 n is the lambda max for nitrophenol at a high pH

How is the initial velocity of an enzyme reaction determined?

the rate or slope of the linear portion of the curve during early time interval

What contributes to the decline in reaction rate?

depletion of substrate or cofactors

What is the purpose of the standard curve used in this experiment?

determine relationship bet OD410 (absorbance) & concentration of nitrophenol

Describe how the nitrophenol standard curve is generated in this experiment. What parameters are used as X- axis and Y- axis?

x = nitrophenol concentration (nmol/ml)

y= OD410 absorbance