Bio Lab Exam II

SYBR green dye

- binds to DNA - fluoresces under UV!

Put the steps of PCR in order

1. Assemble reaction with template DNA and master mix. 2. Denature 3. Anneal 4. Extend

The central dogma of molecular biology. Put the steps in order: the molecules and the processes that create one from the other.

1. DNA 2. Transcription 3. mRNA 4. Translation 5. Protein

Put the following steps of a sequencing experiment in order from start to finish.

1. Extract DNA from the organism/cells. 2. Run PCR so that there are many copies of the sequence of interest. 3. Send samples for sequencing. 4. Analyze the sequence using tools like BLAST.

What are the factors responsible for movement of gel electrophoresis

1. size and shape of DNA fragments 2. density of the gel matrix 3. amount of buffer solution 4. the strength of the electric field (we used 127 V, low voltage to help in migration) 5. electrical charge on the fragment

It is frequently necessary to perform dilutions of a stock solution in the laboratory. If you were instructed to make a 1:10 dilution of a stock solution to arrive at a final volume of 100, how much stock solution would you need to pipet?

10

Cells from an organism were observed under the microscope and the cell cycle status of each cell was recorded as in the table below. Phase # cells % of cells Prophase 79 10.4% Total 757 Approximately how many hours are these cells in prophase?

2.5

How do you calculate the amount of DNA produced?

2^n, with n being the number of cycles

How many cycles did we perform in lab? for PCR

30 cycles

There are 117 offspring from a cross of AaBb x AaBb. If Mendelian ratios are followed, how many offspring would you expect to have recessive phenotypes for both traits?

7

If you allow a heterozygous tall plant that is heterozygous for purple flowers to self-pollinate, how many tall and purple plants would you expect in the F2 generation if you sample 16 individuals? Both tall and purple flowers are dominant.

9

How will the bands on the gel be visualized?

A UV light will make the fluorescent dye attached to the DNA glow.

You are preparing to set up a Punnett square for the cross, AaBb x aabb. The gametes for the underlined parent would be:

AB, Ab, aB, ab

This laboratory experiment uses E. coli strain K-12. While a number of E. coli strains are pathogenic to humans, E. coli is also a natural (and integral) part of the human microbiome in the colon in healthy people. What is unique about strain the strain of K-12 we use that makes it much safer to use in the laboratory?

All of the answers are correct.

Which of the following are true statements regarding safety in this week's lab?

Arrive wearing close-toed shoes.

Which of these statements are good examples of following lab safety protocols?

Asking the TA if you can step outside to get a drink of water.

This week's lab makes frequent use of a bioinformatic tool called BLAST. What is the acronym BLAST short for?

Basic Local Alignment Search Tool

Because the Arabidopsis are transgenic (they have had genetic material added/modified), what is the proper disposal procedure?

Biohazard disposal

Match the function to the name of the lab equipment. Centrifuge Thermocycler Micropipette Chelex beads

Centrifuge: Spins at a high speed to separate things by density (denser stuff moves lower in the tube). Thermocycler: Can be programmed to keep samples/reactions at specific temperatures for specific lengths of time. Micropipette: Measures and transfers very specific, usually small, volumes of fluid accurately. Chelex beads: Bind metal ions so they can be removed from the sample.

Match the PCR component with its definition. DNA template Primers Master mix dNTPs

DNA template: The original DNA that you want to copy using PCR. Primers: Small sections of DNA that match ends of the template so it can be copied. Master mix: The solution that contains all the components--including enzymes, nucleic acids, and ions--to build new DNA. dNTPs: Individual nucleic acids (A, T, C, and G) that will be the building blocks when new strands of DNA are made.

Which genotypes are possible from a cross of parents with the genotypes Dd x DD?

Dd DD

Match the definitions with the PCR terminology. Denaturation Annealing Extension Thermocycler

Denaturation: The DNA template is heated so the strands separate. Annealing: The reaction is cooled so the primers can stick to the DNA template. Extension: The temperature is changed to the ideal temperature for Taq polymerase to build (synthesize) a new strand based on the template. Thermocycler: The machine that controls the temperature of the reaction and has been programmed to move through the heating and cooling steps.

Which type of microscope is best for observing presence/absence of trichomes?

Dissecting

Which of the following values show how closely matched your sequence is to potential matches from the database?

E-value max score identity score

Which of the following are not allowed in lab?

Eating food Chewing gum Drinking water (or anything else) Applying cosmetics (including lip balm)

Identify which of the following statements are true and which are false. Electrophoresis works by running an electrical charge through the gel. DNA has a negative charge. Because of its charge, DNA will move towards the negatively charged side of the gel. All DNA will move through the gel at the exact same rate.

Electrophoresis works by running an electrical charge through the gel. B. TRUE DNA has a negative charge. B. TRUE Because of its charge, DNA will move towards the negatively charged side of the gel. A. false All DNA will move through the gel at the exact same rate. A. false

This week's procedures will include:

Examining plants under blue light to look for GFP. Examining plants under the dissecting scope to look for trichomes.

Gel [a] uses an electrical charge to move [b] through a gel matrix made of [c]. This causes fragments to be separated by [d].

Gel electrophoresis uses an electrical charge to move DNA through a gel matrix made of agarose. This causes fragments to be separated by size.

Which statements about genetics and heredity are TRUE?

Genotype refers to the alleles an individual possesses The genotypic and phenotypic frequencies in a cross between AaBb x AaBb are NOT the same.

Based on the phenotypes of the P generation and F1 generation, what are possible phenotypes in the F2 generation?

Glabrous, no GFP Trichomes, GFP positive Trichomes, no GFP Glabrous, GFP positive

When performing this laboratory exercise, which of the following personal protective equipement (PPE) is required?

Gloves Closed-toe shoes Goggles

What is the correct disposal location for each of the following waste items? Answer choices may be used once, more than once, or not at all. Gloves Tips Tubes Agarose gels Paper towel

Gloves Trashcan Tips Tip bucket Tubes Tip bucket Agarose gels Biohazard waste Paper towel Trashcan

If the graph represents the change in β-galactosidase production (Y-axis) in E.coli cells over time (X-axis), which of the following is true?

I- The amount of allolactose is highest at B and E (the peak points of the graph) III- The amount of active repressor is highest at D and G (the lowest points of the graph)

Which of the following are common uses for BLAST?

Identifying an organism Determining the function of an unknown gene Comparing my sequence to many thousands of others to try to gain insight into the ancestry of the gene.

Which of the following are true statements about Arabidopsis?

It is a model organism. Arabidopsis is easy to grow in a lab. It is possible to create plants that carry genes not normally found in Arabidopsis.

Why do the cheek cells need to be lysed before setting up PCR?

Lysing breaks open the cells so the DNA is accessible.

What is the purpose of PCR?

Make many, many copies of a specific region of DNA

Which of the following will be components in your PCR mixture?

Master mix Human DNA

Which of the following are options for comparing sequences on NCBI BLAST?

Nucleotide BLAST: nucleotide sequence compared to nucleotide sequence tblastn: protein sequence compared to translated nucleotide sequence blastx: translated nucleotide sequence compared to protein sequence Protein BLAST: protein sequence compared to protein sequence

What does PCR stand for?

Polymerase chain reaction

Of the following, which are sequences you can search the databases of GenBank with?

Protein DNA

Complete the sentences by matching. Shoes must be Clothing and jewelry Long hair To avoid transferring chemicals to sensitive areas,

Shoes must be close-toed and cover most of your foot. Clothing and jewelry should not dangle. Long hair should be tied back. To avoid transferring chemicals to sensitive areas, avoid touching your face and eyes in lab.

What does STR stand for?

Short tandem repeats

What are you trying to amplify using PCR in this experiment?

Short tandem repeats from specific genes

The Arabidopsis will be scored based on presence or absence of two traits. A plant that is [a] lacks structures called [b] on its leaves. Plants positive for [c] will glow green under a special light.

The Arabidopsis will be scored based on presence or absence of two traits. A plant that is glabrous lacks structures called trichomes on its leaves. Plants positive for GFP (green fluorescent protein) will glow green under a special light.

The [b] assay can be used to determine expression of [l] under different environmental conditions. If the gene is expressed and the [e] is present, [o] will be broken down and create a [y] pigment.

The B-gal assay can be used to determine expression of LacZ under different environmental conditions. If the gene is expressed and the enzyme is present, ONPG will be broken down and create a yellow pigment.

Lane 4 does not show any visible DNA. Which of the following are plausible reasons there is nothing visible in this lane?

The DNA sample did not contain the region PCR was supposed to amplify so there is no DNA fragment to visualize. The lane was accidentally skipped when loading the gel. PCR of that sample did not work so there is no DNA fragment to be visualized.

What is bioinformatics?

The application of computer technology and associated software to biological data.

At the conclusion of Gene Expression and Regulation experiment, all used culture materials should be placed in what location?

The liquid waste disposal container on the side bench.

What did the master mix consist of?

The master mix contains buffer, loading dye, deoxynucleotides, three pairs of primers, Taq polymerase, and water.

Which of the following are TRUE statements about this week's procedures?

The plates can grow contaminants in addition to plants so be careful how long the plate is left open. The seeds come from the F1 plants and are already fertilized.

What happens during Denaturation of PCR? what temperature?

The reaction temperature is increased to 95 °C, which melts (disrupts the hydrogen bonds between complementary bases) all dsDNA into single-stranded DNA (ssDNA).

What is Extension in PCR? What temperature?

The temperature is increased to 72 °C, which is optimum for DNA polymerase activity to allow the hybridized primers to be extended.

What happens during Annealing of PCR? What temperature

The temperature is lowered to approximately 5 °C below the melting temperature (Tm) of the primers (often 50-55 °C) to promote primer binding to the template.

After sequencing an unknown gene from a mouse, you BLAST it to try to figure out the function of the gene. You use a blastx, which compares the translated nucleotide sequence to protein sequences. The following are your results. What do they mean? The identity score is 97%.

The unknown gene in the mouse probably encodes a protein similar to the paired box 2 (pax2) protein.

Select the safety statements which apply to this lab.

There are reagents and supplies that require disposal in biohazardous waste. Loose/dangling clothing/accessories should be removed or contained so they will not fall into experiments. Arrive at lab already wearing close-toed shoes. Goggles must be on and covering eyes when any experiments are being done in the lab.

This week, two generations of plants will be observed: the [a] that were crossed to create the first generation and those offspring, called the [b] generation. The seeds that you will plate are the [c] generation.

This week, two generations of plants will be observed: the parents that were crossed to create the first generation and those offspring, called the F1 generation. The seeds that you will plate are the F2 generation.

To examine the [a] plants for the [b] of interest, a dissecting scope will be used to look for presence of [c] and a [d] will be used to look for GFP.

To examine the F2 plants for the phenotypes of interest, a dissecting scope will be used to look for presence of trichomes and a blue light will be used to look for GFP.

Based on the phenotypes of the parental plants and F1 plants from the Heredity I lab, which phenotypes are dominant?

Trichomes GFP positive

When migrating through the [a], [b] fragments of [c] can move quickly so will be found [d] from/to the wells than [e] fragments.

When migrating through the agarose, small fragments of DNA can move quickly so will be found further from/to the wells than large fragments.

When you arrive at lab, you need to be wearing [a] shoes. When it is time to start experiments, you must wear [b] and, to keep you safe, they must remain over your [c] while [d] in the lab is doing experiments.

When you arrive at lab, you need to be wearing close-toed shoes. When it is time to start experiments, you must wear goggles and, to keep you safe, they must remain over your eyes while anyone in the lab is doing experiments.

Why is PCR required before running the DNA on a gel?

Without PCR, there would be too little of the DNA region of interest to see it on the gel.

[x] uses a/an [y] called Taq polymerase to create [z] of copies of a specific region of [w].

[PCR] uses a/an [enzyme] called Taq polymerase to create [thousands] of copies of a specific region of [DNA].

In a prokaryotic operon, the promoter functions to

bind the RNA polymerase.

In the lac operon, the operator functions to

bind the repressor and prevent lac gene expression.

Function of Chelex resin

binds ions that inhibit taq polymerase

In the lac operon, the repressor

binds to allolactose and becomes inactive, allowing lac gene expression to occur.

This cell is in which phase?

cytokinesis

What was the purpose of the loading dye

gives color

While experiments are being conducted in the lab, which of the following must be worn?

goggles, gloves, closed toed shoes

All used pipet tips and microcentrifuge tubes should be disposed of

in the waste bag/bucket on my bench.

Select the end of the gel which has a positive charge.

it's the side opposite the wells (farthest from)

Based on the DNA ladder in lane 1, select the band containing DNA that is 700 bp long.

looking to the right of the DNA ladder, chose the one that was aligned with the 700 bp

this cell is in which phase:

metaphase

In animals, what type of sequence is commonly used to construct a phylogeny and determine ancestry?

mitochondrial DNA

In relation to the wells in gel electrophoresis, where is the positive charge and where is the negative charge

positive charge is opposite of the wells. negative charge is on the side closest to the wells.

If you run different genes in PCR, what are the different components of the master mix?

primers, dNTPs

This cell is in which phase?

prophase

When heterozygous tall plants with purple flowers self pollinate to give rise to tall and dwarf plants with purple flowers, it demonstrates the following:

segregation of alleles

When observing plants, trichomes are

small hairs projecting from the leaves

The ____ the fragment, the farther and faster it will travel

smaller

What was the enzyme used in PCR amplification

taq polymerase because it doesn't denature in high temperatures because it is heat resistant (thermophilic)

Based on the gel image below, which lanes contain samples with DNA fragments of the same size?

the answer to this was the fragments that were at the same band length (had traveled the same distance)

Why did we use multiple STRs?

we used 3 different STRs to correctly identify the perpetrator

How can you differentiate if something is homozygous or heterozygous when looking at STRs?

you have to look at 4 nucleotides. if STR are the same size, as in, if there are the same number of STRs, then it's homozygous. If not, heterozygous.