Essentials in Molecular Genetics BIOL 3222 Quiz Preps

What result do you expect for the following on LBAX plates: 10 uL of the QuickChange reaction with Dpn1 treatment:

Blue colonies

In the module video, what color did the bacterial suspension turn when the P2 buffer was added?

Blue.

Make sure you understand the purpose of the different control plates you use in this experiment.

Blue: pUC119 Green: pUC-tet White: pUC-CAP Orange: pACYC184 Controls show what positives for active Lac-Z' genes look like along with controls for antibiotic resistances

How was the allelic difference in this gene among students in the class recognized?

By cutting the PCR fragment with HaeIII

What plasmid is the source of the DNA that will be used as the insert DNA in your cloning experiment?

pACYC184

Which of the following would be a consequence of not following the safety guidelines:

lose all the grade points for that day's exercise

In the pUC119 vector, what does the LacP component allow the investigator to do with an inserted gene?

Express the inserted gene.

What are the four basic steps in a transformation experiment and what is happening in each step?

(1) Preincubation → neutralize the negatively charged lipid phosphates (2) Incubation → Continuing neutralization and congeal the cell membrane, stabilizing the distribution of charged phosphates with CaCl2 (3) Heat Shock → rapid temperature change creating a thermal imbalance and DNA bound to the surface of the cell is taken up (4) Recovery → Cells are suspended in broth at a comfortable temperature so they can recover, amplify the plasmid and express the genes.

Assuming a random sequence of DNA, what is the probability of cleavage for HindIII which has the recognition site: AAGCTT?

(1/4) ^6

Transformation Efficiency

(Colonies observed) / (Mass of plasmid DNA spread) = Transformation Efficiency (colonies/ug)

When you look at the genotype of DH5alpha (see table 3 in lab manual), what genes are deleted so that make the insertional activation system work in your experiment?

(Lac ZYA-argF) U169 deleted allows for LacZ' insertional inactivation in vectors possessing expressible LacZ' alpha-peptide when transformants are grown on Xgal media

Fraction of total cell suspension

(Volume of suspension spread)/(Total volume of cell suspension) = fraction spread on plate (No units, can be made into a percent)

If you are given a plasmid map, be sure that you are able to determine which restriction enzymes you would use to make a recombinant DNA molecule that has certain antibiotic resistance genes.

Cut sites should not interfere with the desired genes. If two genes need to be in the same fragment, do not cut in between but instead cut to open the plasmid so both genes are connected.

Why is the charge to mass ratio of DNA relatively constant during electrophoresis?

DNA is negatively charged and there is one phosphate per base in the DNA sequence.

Why is the charge to mass ratio of DNA relatively constant during electrophoresis? Why is this important in the electrophoretic separation of DNA separation of DNA fragments?

DNA is negatively charged due to its phosphate residues in the backbone and because there is one phosphate per base in the DNA sequence, the charge-to-mass ratio of DNA is relatively constant. This results in a constant force per base pair being applied to each molecule. Because the agarose resists the movement of DNA molecules proportionally to the size of the molecule, a separation of the DNA molecules in the gel during electrophoresis occurs that is proportional to their length. Short DNA fragments will migrate faster and further in the gel.

What polymerase is used in this reaction and why? What is the error rate of the polymerase used in this reaction and why is this acceptable? Why is the QuikChange method not a polymerase chain reaction?

DNA polymerase Pfu - It has a proof reading ability and therefore makes fewer mistakes which results in more successful copies This error rate is acceptable because this reaction is not a PCR reaction and requires more more accuracy for making copies of the originals. A lower error rate is needed so that the few pieces copied are accurate since fewer copies are made. QuikChange is not a PCR reaction because it does not make copies of copies, instead it only makes copies of the original strand.

How did his viewpoint contrast with that of earlier biologists?

Biological forms are static and unchanging from their created forms

If you carried out an experiment with a set of primers that flanked the entire length of the wild type Adh gene (1800 base pairs), what PCR fragment size would you expect if you used Adh F mutant DNA as a template?

1837 bases

According to the module video, what was the purpose of the pUC19 in the transformation reaction shown?

a control

Know the ranges of the micropipetters you will be using and which of the four you would use for a specific volume.

0.5 - 10 uL 5 - 50 uL 20 - 200 uL 100 - 1000 uL Never exceed the largest or smallest value for accuracy and protection of the micropipettes

What are four important properties of PCR primers?

1) complementary to the boundaries of the target DNA sequence 2) the 3' end of the primers must be oriented towards each other 3) must have a melting temp of 50-60 degrees 4) Specificity to flanking regions of target DNA sequence

What are the five basic tastes in mammals?

1. Bitter 2. Sweet 3. Salty 4. Sour 5. Umami

What are four areas of cell preparation that seem to be important for generating maximum competency in cells for transformation?

1. Cell growth 2. Treatment times 3. Treatment temperatures 4. Purity of reagents

What are three things you should do at the end of every lab?

1. Clean and put away equipment 2. Wash hands 3. Inspection

What are two important aspects of the principle of containment of recombinant DNA in the laboratory? (What are two aspects of the principle of "biological containment")?

1. Cloning DNA should only be performed in strains of weakened bacteria that have a very low probability of survival in the natural environment ("weakened host organisms") 2. DNA should be recombined only into vectors that not readily transmit to other organisms ("low transmissibility vectors")

What are the three conformations that double-stranded DNA can adopt and how do they migrate differently in a gel. Which form migrates true to its size? Why do the other two forms of double stranded DNA not migrate true to their size?

1. Closed circular: may migrate faster if negatively supercoiled or migrate slower if positively supercoiled negative supercoil --> faster positive supercoil --> slower 2. Nicked circular: migrates slower than linear because of a break in covalent connections between phosphate groups and the ribose sugar residues in the backbone causing the supercoil to relax --> slower than linear, still chunky and hard to move 3. Linear: migrates true to size --> true to size, moves through gel like a snake in a forest

What are the four properties that affect the migration of DNA fragments through agarose gel matrices?

1. DNA mass 2. Concentration of the agarose in the gel 3. Conformation of the DNA 4. The amount of current applied through the gel

What are the three steps in the QuikChange method and what is actually happening during each step?

1. Denaturation: high temperature denatures ds DNA 2. Annealing: Primers form complex with complementary positions on plasmid strands - temperature for annealing based on the melting temperature of the primer 3. DNA polymerase elongates the plasmid - copies of copies made - significantly less product than PCR, fewer mutations

What are the three elements of a single cycle of PCR?

1. Denature db DNA: 95 degree Celsius 2. Anneal primers to template DNA: 55 degree Celsius 3. DNA synthesis by Taq polymerase: 72 degree Celsius

Why is double stranded DNA especially suited to size separation in electrophoresis?

1. Double stranded DNA has a very uniform structure. 2. At any given pH, there is a constant negative charge for every nucleotide in the molecule.

If you carried out an experiment with a set of primers that flanked the entire length of the wild type Adh gene (1800 base pairs), what PCR fragment size would you expect if you used Adh-fn23 mutant DNA as a template?

1766 bases

What are the five steps in a molecular cloning experiment?

1. Isolate the DNA of interest and prep ends for ligation into the vector. 2. Cut vector DNA with same restriction endonucleases 3. Covalently attach desired DNA fragment into the restricted vector. Ligate. 4. Transformation of bacteria with new recombinant plasmid DNA. 5. Identify clone containing vector. Plate on Amp, Tet, Xgal plate. Select white colonies.

What are three advantages of using Ready-to-Go PCR beads and what do they contain?

1. Provides reagents 2. Decreases the pipetting steps 3. Increases reproducibility (buffer dNTP's and recombinant puReTaq DNA polymerase.)

What are the two chief requirements of a cloning vector?

1. Replicate autonomously 2. Be able to identify cells that have it

How is taste recognition mediated in mammals?

1. Taste molecules bind to a specific receptor on the surface of the taste cell 2. Taste cell generates a nervous impulse which is interpreted by the brain

Be familiar with the three different ways bacteria can acquire DNA from the "environment" (or other bacterial cells).

1. Transformation: taking DNA from the environment 2. Conjugation: bacteria to bacteria from sex pilli (connecting tube) 3. Transduction: DNA from a bacteriophage --> infects bacteria with DNA

What are three features of our experimental design that insure high fidelity DNA synthesis? After the in vitro mutagenesis reaction, how was the original template destroyed and how did you prove that it was destroyed?

1. Use of high fidelity Pfu polymerase 2. Only using original plasmid as template to make copies 3. Dpn1 digestion of the parental strand The original template is destroyed by Dpn1 digestion. We ensured that this occurred through plating and analysis against the undigested plasmid on X-gal and gel analysis (less bright bands)

What are four advantages in the QuikChange method with regard to making a site-directed mutation?

1. Uses 2 complementary olives that encode the mutation in both strands of the new DNA 2. Uses normal ds plasmid DNA 3. Uses "high fidelity" thermostable DNA polymerase and a thermocycler to amplify DNA 4. Selects in vitro against the non-mutated plasmid by Dpn1 digestion of methylated DNA

What are three ways of mishandling a micropipetter that could lead to liquid entering the barrel of the device?

1. Using a pipette without a disposable tip 2. Tipping the pipette up so that the barrel is level or higher than the handle 3. Laying down the pipette with liquid in the tip

What are the four specific sequences a cloning vector should contain and what is the function of each?

1. a replicon - direct the replication of DNA 2. a selectable gene or gene product - allows you to distinguish between cells that took up the vector 3. unique cleavage sites for restriction enzymes - makes the reproducible insertion of "foreign" DNA into specific sites in the plasmid possible 4. suitable expression control elements - allow the discrimination of cells containing vectors that have foreign DNA cloned into them from cells that were transformed with only the original vector WITHOUT foreign DNA

What were three important aspects of Mendel's genetics that T. H. Morgan and his colleagues discovered as they induced mutations and carried out genetic crosses in Drosophila melanogaster?

1. physically located on chromosomes 2. Can be mapped with "crossing over" 3. New mutations occur simultaneously every generation

Which of the following micropipetters use a blue tip?

100ul to 1000ul

How many bacterial cells can grow from a single bacterial cell in a simple overnight conditions under optimal growth conditions?

10^7 - 10^8

How many bacterial cells can grow from a single bacterial cell in a simple overnight conditions under optimal growth conditions?

10^7 to 10^8 bacteria will optimally grow overnight

According to the module video, how many bases should flank the sides of the mutation in the primers that will direct the base change in the QuikChange reaction?

12 on each side

If you were starting with 100 molecules of DNA in a PCR reaction, how much DNA would you expect to have after 30 cycles of amplification?

1x10^9

How many genes encode receptors for bitter taste in mammals?

30 bitter genes

The E. coli genome has approximately how many genes?

4,300 genes

You are using these two plasmids to make a recombinant DNA molecule. You want to make a molecule that has both ampR and kanR. How large in base pairs will be your desired recombinant DNA molecule?

5630 base pairs. 3755 bp from pAMP and 1875 bp from pKAN.

The study of molecular genetics in Drosophila is relevant to human disease or genetic conditions because:

75% of known human disease genes have a recognizable match in the genome of fruit flies

How are Primers 1 and 2 used to make the nucleotide change to restore B-gal activity to pWS4.5?

A correction of the nonsense stop mutation found in the B-gal gene of the pWS4.5 plasmid will be performed through "correcting" primers Primers 1 and 2 have complementary olives that encode the correcting mutation in both strands

Describe an experiment that you could conduct using PCR that would allow you to distinguish both Adh-F and Adh-fn23 from the wild type Adh gene and from one another (assume the wild type Adh gene is 1800 base pairs long). What new elements of the experiment would you use?

A different set of primers

What is insertional inactivation and how is that used with the cloning vector, pUC119?

A foreign DNA fragment is inserted into a restriction site within a gene for antibiotic resistance making it nonfunctional. In pUC119 has ampicillin resistance and it can lose it through insertional inactivation.

According to the module video, who or what is J Screen?

A genetic screening service for parents of Ashkenazi Jewish descent

What does the term "miniprep" mean in the context of this laboratory?

A plasmid DNA preparation that is expected to yield microorganisms from a couple of mL of cell culture.

What is a Multiple Cloning Site and where is it usually positioned in a cloning vector? What advantage does a MCS sequence give an investigator who wants to clone a particular gene into a cloning vector?

A sequence of unique restriction sites that are genetically engineered into the vector in a very short region of DNA. They are placed near the very beginning of the protein coding sequence. They are advantageous because they allow you to add restriction sites and extra DNA into the protein coding region of a gene without functionally inactivating the gene product.

What is the molecular explanation for a strong tasting ability for PTC?

A specific combination of single nucleotide polymorphisms (SNPs) that constitute a halpotype associated with the strong tasting ability

Define "electrophoresis."

A technique that is used to separate macromolecules based on their size.

In the procedure you used in the QIAspin Miniprep: a. How were the cells lysed? b. What were the three effects of adding Potassium acetate solution to the cellular lysate? c. Why is it important NOT to vortex the neutralized lysate prior to microfuging it? d. After this microfuge spin, what is contained in the supernatent and what is contained in the precipitate? e. When the supernatent is poured over the QIA spin column, what binds to the silica matrix? f. How is the plasmid DNA released from the column?

A) add 250 uL of buffer P2 (NaOH) to the cell solution and inverting it 6 times. B) Effects of adding Potassium Acetate to cell lysate: 1. Potassium acetate raises the salt concentration of the cell lysate to a very high value 2. Neutralize pH 3. Removes detergent used to solubilize the membrane of the cell C) Vortexing the neutralized lysate before microfuging would shear long chromosomal DNA into short fragments and contaminate the plasmid DNA. D) Post-microfuging: Supernatant contains plasmid DNA Precipitate contains cellular debris and chromosomal DNA E) Plasmid DNA binds to the silica matrix when the supernatant is poured over the QIA spin column. F) DNA is released from the column by adding small volumes of low ionic strength buffer that elutes the plasmid DNA and releases it. A final spin will remove the plasmid and deposit it purely and cleanly into a new tube.

Assuming a random sequence of DNA, calculate the probability of cleavage by the restriction enzyme HindIII which has the recognition sequence: A/AGCTT. If the E.coli genome is 46 kb in length, how many HindIII site would you predict to find in this genome?

A/AGCTT probability: (1/4) ^5 = 1/1024 E. coli genome is 46kb/1024 ~ 44.921 so 45 HindIII sites are expected to be found.

What is the normal function(s) of E. coli in the human colon (in both the absence and presence of O2)?

Absence of O2: - What Ferment sugars into lactic acid Presence of O2: - Waste processing and nutrient absorption - Oxidize sugars to CO2 and H20 - Vitamin K production

Total volume

Add all of the volumes used to prepare the solution. Volume of: - cell suspension - DNA added (pUC119/Lig) - LB broth (uL)

In lab, you observed that Adh-F homozygote flies were able to tolerate alcohol better than Adh-fn23 homozygote flies. Given the molecular nature of each mutation, explain the difference in alcohol tolerance between these mutants.

Adh-F has a 37 base pair insertion and there is potential for some gene products to still create some functional proteins. Adh-fn23 removes the stop codon that results in a nonfunctional protein. The Adh-fn23 flies are more susceptible to alcohol without a protective functional protein while Adh-F has some protection with a compromised functional protein from alcohol intoxication.

During what step in the plasmid prep is plasmid DNA separated from chromosomal DNA in a single tube?

After the N3 buffer has been mixed in and centrifuged, chromosomal DNA is collected at the bottom as pellet. The plasmid DNA is separated when the supernatant of that centrifugation is transferred into the QIA prep spin column.

What is agarose and where does it come from?

Agarose is a mixture of long chains of saccharides. It comes from red algae.

What step in the process of alcohol metabolism is catalyzed by the enzyme Alcohol Dehydrogenase in HUMANS and in what organ does this conversion take place?

Alcohol Dehydrogenase hydrolyzes ethanol into acetaldehyde In the liver

Why is the QuikChange method not a polymerase chain reaction?

Because the primers are complementary to one another

What are the mechanisms of action of ampicillin resistance and tetracycline resistance?

Ampicillin Resistance: have a gene that encodes an enzyme known as beta-lactamase, binding to the beta-lactam structure of ampicillin and hydrolyzes the ring, destroying the cell wall structural mimicry - This allows the cell to carry on and build new cell wall. Tetracycline Resistance: Cells that are tetracycline resistant have an efflux pump ATP-driven transporter that binds the tetracycline and translocates it out of the cell.

What are the mechanisms of action of ampicillin and tetracycline?

Ampicillin: irreversibly binds to the transpeptidase which inhibits it from synthesizing part of the peptidoglycan cell wall - Causes the cell to burst Tetracycline: binds to the 30s ribosomal subunit, inhibits the cells protein synthesis by stopping the aminoacyl-tRNA binding at the A site of the ribosome

A plasmid containing an ampicillin resistant gene (ampR) produces?

An enzyme that modifies the active chemical structure, rendering it useless

Which the four specific sequences of a cloning vector described below provides the basis for selecting for or against cells that were successfully transformed?

Antibiotic resistance gene.

What selection method do you think will be used to recognize the presence of this fragment?

Antibiotic resistance of Amp and Tet to test fragment uptake.

How could a mutation in the human MDR1 gene affect the cancer treatment plan for a patient who contains a constitutively active MDR1 protein in their tumor cells?

Any cancer cell over-expressing the pump will cause the cell to survive treatment therapy to many chemotherapeutics because the medicine is pumped out of the cells before being able to destroy the cancer

In the safety principle of "biological containment," what is being contained within the laboratory?

Any recombinant DNA that are used or synthesized in the experiment

How are cells made "chemically" competent to take up DNA?

Bacterial cells are grown to log phase and harvested and the cells are treated with calcium chloride which neutralizes the charge of the DNA. Cells are then heat shocked to crack the cell membrane to allow DNA to enter. The cells then incubate and recover to replicate the newly added DNA.

Why is culturing bacteria in the laboratory an essential part of any molecular biological or molecular genetics experiment?

Bacterial cultures are used to quickly and accurately amplify the quantity of any gene or gene product that the bacterium contains, allowing us to better study and isolate those genes

What is the difference between bacteriocides and bacteriostats?

Bacteriocides: Kill the bacteria Bacteriostats: Inhibit the growth of the bacteria

Why is Taq Polymerase used in the PCR reaction?

Because it has an enzymatic optimum temp of 70-80 degrees Celsius and can withstand temps above 90 for reasonably long periods of time, so it doesn't have to be replaced after each cycle

In a PCR reaction, why is only the DNA between the primers amplified in the majority of the cases after 30 cycles?

Because the 5' end of the primers define the end of the first products

In a PCR reaction, why is only the DNA between the primers amplified in the majority of cases after 30 cycles?

Because the 5' end of the primers define the end of the first products The polymerase can't exceed the bordering primers so only the desired DNA is amplified

Why are the new copies from the pWS4.5 template NOT used as template in subsequent reactions?

Because the new copies of pWS4.5 are linear and not continuous circular plasmids

Why are the new copies from the pWS4.5 template NOT used as template in subsequent reactions?

Because the new copies of pWS4.5 are linear and not continuous circular plasmids like the template

What is the PCR annealing temperature (Ta) for the following sequence: 5' - CCATGTTTAGGCCTAGCA -3'

C: 5 Ta = 4(C+G) + 2(A+T) - 5 G: 4 = 4*(9) + 2*(9) - 5 A: 4 = 36 + 18 - 5 T: 5. Ta = 49 C

What is used to make bacterial cells "chemically" competent to take up DNA?

CaCl2

What is agarose?

Carbohydrate (starch)

The target of tetracycline are cells in what stage?

Cells that are actively involved in translation

What is the normal function of MDR genes in the cell?

Cellular membrane proteins that export chemicals and toxins out of the cell

Name one traditional method of making a genetic mutations. Why would an investigator want to make a site-directed mutagen? What advantage does it provide over a traditional method of mutagenesis?

Chemical mutagens and radiation Site-mutagenesis allows for more control of which gene is mutated. It allows for specific phenotypic mutations or functional mutations for research.

Complete Digestion of DNA

Cleaving all recognition sites completely. Expected number of bands on gel. No smear.

In the module video, when a DNA profile is generated from material found at a crime scene, but no suspects have been identified, what do investigators do next?

Compare DNA profile to those stored on a database called CODIS.

What does "competence" mean and what is the difference between "artificial" and "natural" competence?

Competence: the ability for bacteria to take up DNA from the environment Artificial competence: Bacteria is treated and the cells are treated with certain reagents and procedures to increase their competency in a lab Natural competence: Bacteria that can control the ability to take up DNA and use it in times of high stress

What specific feature of the behavior of nucleotide bases of the DNA was critical to determining the correct structure of DNA by Watson and Crick?

Complementary base pairing

How are bacteria-containing materials normally disposed of according to federal law:

Contaminated materials can be disposed of in a biohazard bag that is thrown into the trash after autoclaving.

What step in the process of alcohol metabolism is catalyzed by the enzyme Alcohol Dehydrogenase in humans and in what organ does this conversion take place?

Conversion of ethanol to acetaldehyde In the liver

In what form is your plasmid DNA (pUC119) when it is eluted from the spin column?

Covalently closed, supercoiled DNA.

How will you covalently link the insert DNA to the cloning vector?

Covalently link using T4 DNA Ligase with optimal conditions.

If you wanted to resolve DNA fragments that were larger, how would you change the concentration of the agarose in your gel?

Decrease the agarose concentration

In the module video, what research were scientists doing in the Biological Safety Level 3 lab at St. Jude's in Memphis, TN?

Developing vaccines against H5N1

What is the proper location for contaminated pipettes or pipette tips when you are finished with them?

Discard beaker or biohazard bin

Why is EDTA used often in biochemistry and molecular biology?

EDTA inactivates enzymes that would otherwise breakdown DNA by removing metal ions, particularly Mg2+, from the enzymes.

What discovery was instrumental in the birth of the field of molecular biology?

Double helix structure of DNA

In a plasmid miniprep, when the supernatant, following the lysate step, is poured over the QIA spin column, what binds to the silica matrix?

Double stranded DNA.

You have recovered a transformed bacterial cell that can grow on LBA and LBT, but when you streak this bacteria out on LBAX, you get blue colonies. This cell came from the transformation using ligated plasmid used in this experiment. What is a possible explanation for this observation? How would you test your hypothesis?

Double transformant Run an overnight culture on a gel after cutting with HindIII and EcoRI and expect 3 fragments

After the in vitro mutagenesis reaction, how was the original template destroyed?

Dpn1 digestion

Why is the study of molecular genetics in Drosophila relevant to human disease or genetic conditions?

Drosophila genetics are relevant to human disease and genetic conditions because they have so many alleles of its single Adh gene that suggests that it is a good system to start with in designing a DNA test for alcohol tolerances

What was the first animal genome to be entirely elucidated?

Drosophila melanogaster

What was the first animal genome to be entirely elucidated?

Drosophila melanogaster (fruit fly)

At what point in the QIA plasmid prep procedure is plasmid DNA separated from bacterial DNA and cellular proteins?

During the clearing lysate step

What strains of bacteria are typically used in a lab at Biological Safety Level 1?

E. coli

Why is the E. coli serotype K12, which we use in the laboratory, not pathogenic? What characteristic has it lost and why?

E. coli K12 is nonpathogenic because of colonization outside of the human body for a significant amount of time has caused it to lose the O-antigen that is required for infection O-Antigen is a lipopolysaccharide that is present on the cell surface

What is the size of the bacterial genome of E. coli in base pairs and compare this to the size of the cloning vector pACYC177 in base pairs.

E. coli genome is 4.6 million base pairs Cloning vector pACYC177 is 3941 base pairs

Describe on feature of Drosophila that makes it a great organism for molecular genetics?

Easy to grow Have short life cycles Can be tested in small bottles or virals

If you were going to use this vector to clone a foreign fragment of DNA, what two restriction enzymes would you use to cut the vector, so you could identify a recombinant DNA molecule as ampR AND distinguish it from a reannealed pBR322 that will have both ampR and tetR?

EcoRI and Bam HI. Interrupt the genome of one of the resistance genes so that when plated it will not have the gene for TetR.

If you were going to use this vector to clone a foreign fragment of DNA, what two restriction enzymes would you use to cut the vector, so you could select the recombinant plasmid that has both Amp and Tet resistance?

EcoRI and HindIII. Cut to open the plasmid as to not separate the genes into two separate fragments.

In what form is your plasmid DNA when it is eluted from the column (linear or covalently closed, relaxed or supercoiled?) What protects the plasmid DNA from being damaged (nicked) during purification?

Eluted plasmid DNA is covalently closed and supercoiled. The DNA is bound to the matrix while being purified so very little damage occurs.

What is one chemical danger in this laboratory course and why is it dangerous to you?

EtBr or ethidium bromide. It is dangerous because if exposed it may intercalate between the bases of DNA double helix, causing DNA polymerase mutations during DNA replication, making it a suspected carcinogen

What must be worn at all times in lab, assuming you are wearing closed toed shoes and long pants already?

Safety goggles

You have recovered two LacZ-minus AmpR colonies from your transformation in Lab 7 and they appear as white colonies on a LBAX plate. You test each colony on a LBT plate. Only one of these two colonies grew on the LBT plate (Colony 1) while the other colony did not grow on the LBT plate (Colony 2). If the plasmids from Colony 1 and Colony 2 were purified and digested with Hind III and Eco R1, you would expect Colony 1 to have two restriction fragments (2.7kb and 3.1 kb), while Colony 2 would have two restriction fragments (2.7kb and 1.5kb).

False *I have changed the answer to this question to be False, because Colony 2 would have two fragments: 3.1kb and 1.5kb.*

What is the purpose of having a Bunsen burner (or alcohol burner) producing a flame on the bench top while you are using sterile technique (other than for flaming glassware and pipettes)?

Fire makes an updraft that minimizes the random deposit of microorganisms on sterile surfaces while working

How did you measure alcohol tolerance in the fruit fly mutants you studied in lab?

Fruit flies sucked up alcohol that was placed on a paper square

How was human DNA isolated and extracted from cells in this experiment?

Saline mouthwash to collect cells that were then boiled in a Chelex resin to extract DNA

If given the starting amount of DNA before PCR amplification, could you determine the amount after 30 cycles of PCR?

Grams -> g/mole -> 6.02*10^23 molecules/mole -> 30 cycles

Why is Taq Polymerase used in the PCR reaction?

Heat stable

What is the correct order of the steps in a PCR cycle?

Heat-denaturation > 90 C Annealing Ta: ~ has 50 - 65 C DNA polymerization 72 C Preserve at 4 C

Which enzymes will be used to generate the desired insert fragment? What advantage does cutting with two restriction enzyme offer in a cloning experiment?

HindIII EcoRI Makes matching sticky ends which will ensure that the fragments will fit into each other's overhangs.

How was human DNA isolated and extracted from cells in this experiment?

Human DNA was isolated from cheek cells and extracted by boiling with Chelex resin

How did Charles Darwin revolutionize biology as it relates to Molecular Genetics?

Hypothesizing that material and worldly mechanisms were driving the adaptations of Earth's living beings —> direct investigation by experimentation

What is the purpose of the MCS sequences being located within the LacZ' gene?

It allows for insertional inactivation as a way to show that the gene has been successfully inserted and has therefore deactivated that LacZ' gene.

What is the molecular mechanism by which ampicillin kills bacterial cells?

It blocks the synthesis of the peptidoglycan layer of the cell wall of the E. coli

What is one drawback of using the original Taq polymerase in PCR?

It has a relatively high error rate because it lacks 5'-3' exonuclease activity

What is one drawback of using the original Taq polymerase in PCR?

It has a relatively high error rate of 1 mismatch per 9000

Why is the E. coli serotype K12, which we use in the laboratory, not pathogenic? What characteristic has it lost and why?

It has lost the O-antigen, the lipoprotein associated with pathogenic strains

One reason Drosophila melanogaster is a great model system for molecular genetics is because it contains the same number of genes as are in the human genome

It has many similar genes to those found in humans "True"

Why is Ethidium Bromide used in the staining of DNA (two reasons)?

It intercalates into DNA and fluoresces under UV light, allowing you to view the separated bands.

What is a COS site?

It is a 12 base complementary overhang in the lambda chromosome. It is required for the packaging of the the phage into phage head during virus construction. It enables lambda to linearize or become circularized.

What is bacteriophage lambda and in what two phases can it exist in the bacterial cell? What is a cos site and what does it allow the lambda to do?

It is a virus that targets and infects E. coli. It has both a lytic phase (where it lyses the host cell) and a quasi-stable prophage with its genome incorporated into the host's chromosome (when the host replicates, so does the phage DNA). The cos site is a single stranded 12-base complementary overhand at each end of the double stranded DNA. When cos is annealed after infection, it is ligated by the E. coli DNA ligase.

What observation is the QIAspin Miniprep procedure based on?

It is based on the observation that DNA will tightly bind to "activated silica" in the presence of high salt solutions.

What advantage does site-directed mutagenesis provide over a traditional method of mutagenesis?

It makes the exact mutation that the investigator wants

Who was responsible for the development of PCR and use of Taq polymerase?

Kary Mullis

In the module video, what was reported to typically have more bacteria than a toilet?

Kitchen Sink

In the pUC119 vector, what does the LacP gene allow the investigator to do with an inserted gene?

LacP is positioned upstream of the lacZ gene. Cloning foreign DNA into the MCS of this plasmid will allow transcription of the inserted DNA. It allows for the expression the inserted gene.

Be sure to know the four different phases of the growth curve of bacteria (lag, log, stationary and death) and where they are on a growth curve. You should also know what each phase means.

Lag: Cells adapting to conditions, bacteria maturing and making enzymes Log: Period of cell doubling and exponential growth Stationary: Growth limiting phase, plateau, depletion of essential nutrients, growth rate = death rate Death: Bacteria die due to lack of nutrients, decline phase

Which Biological Safety Level is reserved for biological agents that can cause potentially severe or fatal disease in humans, but for which effective treatments (for example, antibiotics or vaccines) exist?

Level 3

Which Biological Safety Level is reserved for biological agents that can cause potentially severe or fatal disease in humans, but for which effective treatments (i.e. antibiotics, vaccines) exist?

Level 3

What is one potential fire hazard in this laboratory course?

Long hair, sleeves, or scarves when using the Bunsen burner or alcohol lamp

Which of the following is a definition of "molecular cloning"?

Making many copies of a specific DNA sequence.

The tip on your micropipette has an air bubble in it after taking up a specified volume. What could be the problem?

May not be using the correct disposable tip Corrosion could cause the pipette to lose its seal and calibration

What is the difference between "microchemistry" and "sterile technique" and "microbiology techniques"?

Microchemistry: chemical techniques for preparing, handling, and analyzing small quantities of chemical reactions Sterile technique: a system of practices that are used to prevent contamination of bacterial cultures when handling microorganisms Microbiology: Use of specific strains that have optimized genetic backgrounds for experimental purposed

What was an aspect of Mendel's genetics that T. H. Morgan and his students discovered as they carried out genetic crosses in Drosophila melanogaster?

Mendel's hereditary factors were physically located on chromosomes.

When you are setting up a restriction digestion of 1 ug of DNA in a 20ul volume, you are practicing:

Microchemistry

How do you deliver the liquid in the micropipette tip to a new tube?

Move tip of the micropipette to the receiving tube and depress the spring-loaded plunger to the first and then the last stop to ensure complete dispensing

What are the two chief requirements of a cloning vector?

The ability for it to autonomously replicate The necessity for being able to identify amount all of the thousands and thousands of cells in the culture and those cells that actually contain the vector

Which of the following is a property of PCR primers?

Must be "specific" for the flanking regions of target DNA sequence

What is the correct order of the three steps in the QuikChange method?

Mutant strand synthesis, Dpn1 digestion, transformation

What is responsible for denaturing all DNA to single stranded early in the QIA prep procedure?

NaOH

What was responsible for lysing the cells in the QIAspin Miniprep procedure?

NaOH and detergent.

Which of the following is an effect of adding Potassium Acetate to the cellular lysate?

Neutralizes the pH of the lysate.

How do you verify that the desired volume of a solution has been drawn up into the tip of a micropipette?

No bubbles will be present in the tip Volume should remain in the tip and not drip out

According to the module video, is artificial selection the same as making a transgenic GMO?

No.

How do the two plasmids differ from each other: pWS4.5 and pBluescriptIISK?

One has a mutated Lac Z gene (pWS4.5) and the other has a wild type Lac Z gene (pBLU)

What molecular phenotype is expected for a strong taster in this experiment?

Only 177 bp and 44 bp fragments

What molecular phenotype is expected for a non-taster in this experiment?

Only 221 bp fragment

Under optimal conditions, a typical lab strain of E. coli can divide in how many minutes?

Optimal conditions allow E. coli to divide every 20 minutes

What is the most prevalent MDR transporter that is expressed at high levels in cancer cells resistant to chemotherapy?

P-glycoprotein (Pgp)

Know the names and functions of the buffers you used with the QIAprep column and the order in which they are used.

P1 (buffer with RNAse A): Destabilizes the cell membrane and macromolecules and allows for separation from other cell components. DNase inhibitor P2: Cell lysis by potassium acetate. Precipitates other cell components . Plasmid DNA stays in solution. N3: Acetic acid used to neutralize pH and raise salt concentration. guadium chloride to prevent protein folding. PE: Wash buffer with high pH. Causes the plasmid to attach to the column. Ethanol wash. EB: Elution buffer with low pH to detach clean plasmid DNA into a new tube.

How was the allelic difference in this gene among students in the class recognized?

PCR tasting paper for phenotype and by cutting the PCR fragment with HaeIII for genotype

What is PTC? How does it taste to some people? What is the basis for the inability to taste PTC in other people?

PTC: Phenylthiocarbamide, One of 30 SNPs responsible for the ability to taste for bitterness It tastes bitter to some, not at all to others Those who cannot last are recessive for TAS2R38

How do the two plasmids differ from each other: pWS4.5 and pBluescriptIISK? (DNA sequence, protein expression and colony differences).

PWS4.5: non-functional, truncated Lac Z gene, white colonies pBLU: functional Lac Z, hydrolyzes X-gal, blue colonies

What does "competence" mean in relation to bacteria?

The ability to take up foreign DNA.

Which of the following is a feature of our experimental design that ensures high fidelity of DNA synthesis?

PfuUltra polymerase is used with an enhanced proof-reading ability

According to the module video, what bond is formed by DNA ligase?

Phosphodiester bond.

What method of selection will allow you to recognize a plasmid that has the desired insert if you desire to make?

Plate transformed cells on LB with Tetracycline, Ampicillin and X-gal.

You are creating a recombinant plasmid using pUC119 and pACYC184. After cutting both plasmids with EcoR1 and HindIII, you want to use the pUC119 fragment that has the origin of replication, and the fragment of pACYC184 that does not have its origin of replication. What method of selection will allow you to directly recognize and select a recombinant plasmid that is described in this question?

Plate transformed cells on LB with Tetracycline, Ampicillin and X-gal.

If you observed blue colonies on the positive control X-gal plate, but not on the negative control and experimental X-gal plates, what would be your conclusion about the transformation and in vitro mutagenesis?

Positive control: Blue Negative control: White Experimental: White Conclusion: The transformation and in vitro mutagenesis was not successful

Know the definitions of "precision" and "accuracy" as well as "in vitro" and "in vivo". Make sure you can name an experiment that is done "in vitro" and one that is done "in vivo".

Precision: degree of mutual agreement a series of individual measurements Accuracy: degree of conformity of measurement to its actual (true) value In vivo: perform in living organisms - site specific mutagenesis In vitro: in test tubes - isolation and purification of DNA from animals and bacterial sources - restriction digestion of DNA - recombination of restricted DNA fragments -Polymerase chain reaction amplification

How will you prepare this plasmid to receive the insert DNA?

Prep by cutting with Hind III and EcoRI. Combine fragments in a tube with DNA ligase.

What are the basic elements contained in a PCR reaction?

Primers Template DNA Taq polymerase Free dNTPs Buffer

What is the purpose of having a Bunsen burner (or alcohol burner) producing a flame on the bench top while you are using sterile technique (other than for flaming glassware and pipettes)?

Provide an updraft that will prevent mold or bacteria from falling on surfaces in the immediate vicinity of the Bunsen burner.

You are plating four different DNA samples on LBAmp plates: QC-= 10 μl of the QuikChange reaction (no DpnI treatment) QC += 10 μl of the QuikChange reaction with DpnI treatment pWS4.5= 10 μl pWS4.5 plasmid pBLU= 10 μl pBLU plasmid What results (in terms of colonies - blue versus white) do you expect on each plate? What information does each plate give you about your experiment?

QC-: Blue and white colonies, successful reversion of the mutation QC+: Blue colonies only, successful reversion of mutation and successful and complete digestion of original DNA pWS4.5: White colony control plate, confirms that X-gal is fresh and not self-hydrolyzing pBLU: Blue colony control plate, example of functional Lac Z gene

Which of the following is NOT a basic element (ingredient) contained in a PCR reaction?

RNA polymerase

Which of the following conformations of DNA runs slower than its true size in base pairs on the gel?

Relaxed circle

While loading the well of a gel with loading dye, you notice that the dye goes into the well, but then goes right back up into the tip of the micropipette as you are pulling the micropipette out of the well. What could be the problem with your technique and how will you correct it?

Releasing the plunger while the pipette is submerged within the well would pull dye back into the tip - Instead hold the plunger down until the pipette is completely out of the gel

If you wanted to resolve DNA fragments that were larger, how would you change the concentration of the agarose in your gel and why?

Resolve larger fragments by decreasing the agarose concentration to allow for larger pores so the larger DNA can migrate faster. This allows for more resolution of larger fragments.

Partial Digestion of DNA

Restriction Enzyme not completely digested, some cut sites stay intact. More fragments than expected. Blurry bands. Common in supercoiled DNA.

What is the purpose of the MCS sequences being located within the LacZ gene?

So that insertion can be identified as the failure of expression of LacZ.

When you take measures to reduce the chances of bacterial contamination, you are practicing:

Sterile Technique

In the module video, which bacterium was more susceptible to antibiotics?

Streptococcus aureus

Describe the DNA sequence difference between a homozygous nontaster versus a homozygous taster and how this difference will affect the RFLP following HaeIII digestion. In other words, what molecular phenotype is expected for a strong taster, weak taster and a nontaster?

Strong: TT Weak: Tt Non-taster: tt

For E. coli cells, which form of DNA is easiest to use to transform cells (be taken up by the bacterial cell)?

Supercoiled DNA

How could systematic error contribute to lower the accuracy and the precision of your experimental data in this experiment? Can you give examples of systematic error in each context?

Systematic Error: error due to wrongful use of instruments or instrument data - Corrosion in micropipettes doesn't allow the delivery of accurate volumes which would not allow for proper dilutions or accurate experiments

What is the name for the PTC taste receptor?

TAS2R38

What is the name for the PTC taste receptors and when was it identified?

TAS2R38 80 years ago in the 1930's

What is the purpose of the TBE buffer used in both the gel and in the electrophoresis chamber? Why is it important in the electrophoretic separation of DNA fragments?

TBE buffer provides an ionic solution that allows current to flow through the water. This current allows the DNA to move toward the positive pole, separating the fragments.

Know how to calculate the annealing Temperature for PCR from a given primer sequence.

Ta = 4*(G+C) + 2*(A+T) - 5°C

What are cells that are grown to mid-log phase used for and how long does it take for cells to reach mid-log phase in a standard culture?

Takes about 12 hours for mid-log phase. Cells grown to this phase are used to clone and amplify specific genes

Which of the following is NOT in Ready-to-Go PCR beads?

Template DNA

What is the modern concept or premise of pharmacogenetics?

Testing a person's metabolic response to drugs based on their inherited genetic differences

Which of the following is NOT one of the four areas of cell preparation that seem to be important for generating maximum competency in cells for transformation?

Thawing cells at room temperature

The primers used to amplify a region of the Drosophila Adh gene were predicted to detect a change in length for the Adh-F mutation but not for the Adh-fn23 mutation, yet each mutation changed the length of the gene by approximately 30 base pairs. Explain this discrepancy.

The Adh-F mutation has a 37 bp insertion in an untranslated region before the start codon causing a frameshift. There is no change in the number of base pairs in the gene translation region. The Adh-fn23 mutation had a 34 bp deletion in the 3' coding region, it removes the stop codon so the gene cannot stop at the proper location. The deletion affects the number of base pairs in the selected region of observation.

Why do you think you recovered any transformants in Lab 4 if the cells had a lower expected transformation efficiency, compared to the higher transformation efficiency of the cells in Lab 7?

The DNA in Lab 4 was supercoiled whereas the DNA used in Lab 7 was not supercoiled.

You have recovered a transformed bacterial cell that can grow on LBA and LBT, but when you streak this transformed cell out on LBAX, you get blue colonies. This cell came from the transformation using ligated plasmid used in this experiment. What is a possible explanation for this observation?

The cell was transformed with both pUC119 and pACYC184

What genotype of DH5alpha cells allows you to use the insertional inactivation as described in the lab protocol?

The deletion of LacZYA genes.

What is the driving force for electrophoresis and what does it act on?

The driving force is an electric field that acts on the negative phosphate backbone of the DNA. This cause them to move toward the positive end of the electric field.

What specific feature of the behavior of nucleotide bases of the DNA was critical to determining the correct structure of DNA by Watson and Crick?

The feature that nucleotide bases were hydrogen bonded in a specific pattern that created a double helix.

In lab, you observed that Adh-F homozygote flies were able to tolerate alcohol better than Adh-fn23 homozygote flies. Given the molecular nature of each mutation, this difference can be explained by the following:

The fn23 allele makes a truncated protein, while the F allele has its mutation outside of the coding region of the gene

In experiment Lab 7, why did you use 10x greater volume of ligated DNA in the transformation than control plasmid (pUC119 or pACYC184) when adding the DNA to the bacterial cells.

The ligated DNA is less likely to be viable for cell uptake as the ligation does not go to 100% completion so using a larger volume allows for a higher likelihood to for the success in ligated DNA's addition to bacterial cells.

What is the relationship of the movement of a double stranded DNA molecule in an electric field relative to its mass (length in base pairs)?

The movement of a double stranded DNA molecule in an electric field has been found to be inversely proportional to its mass (because there is a constant charge per mass).

What is the natural purpose of restriction endonucleases in bacteria? How then are molecular biologists able to introduce foreign DNA into strains of E. coli?

The natural purpose of restriction endonuclease is a biological defense system to prevent the expression and possible takeover of a cell of one species by DNA that have been produced in a different species. Biologists are able to introduce foreign DNA through plasmids.

Compare the QIAspin Miniprep procedure to the method of preparing plasmid from a bacterial culture in the past. (In other words, what step does it share with the old method and what new improvement has the QIAspin made on the old method).

The new method begins with a typical alkaline lysis of bacterial culture just as in the old method but differs in the method of purification. The release of plasmid DNA from the other cellular materials uses "activated silica" high salt solutions to purify in the new method.

Why is the plasmid DNA found in the supernatent after clearing the lysate, while the chromosomal DNA is found in the pellet?

The plasmid DNA is much lighter and smaller in comparison to the chromosomal DNA.

In the QIA plasmid prep procedure, plasmid DNA is found in the supernatant after the neutralization of the lysate because?

The plasmid DNA remains double strained, so it is soluble.

How are Primers 1 and 2 used to make the nucleotide change to restore B-gal activity to pWS4.5?

The primers each contain one nucleotide change that restores the wild type sequence of the B-gal gene

The primers used to amplify a region of the Drosophila Adh gene were predicted to detect a change in length for the Adh-F mutation but not for the Adh-fn23 mutation, yet each mutation changed the length of the gene by approximately 30 base pairs. This observation can be explained due to:

The primers used in this PCR reaction flanked only the F allele and did not amplify the region containing the fn23 mutation

Be familiar with the definition of molecular cloning.

The process of making many copies of a specific DNA sequence.

What does in vitro mutagenesis mean? Among the different types of in vitro mutagenesis, which one are you using in lab this week?

The production of either random or specific mutations in a piece of cloned DNA. The DNA is then reintroduced into a cell or organism. Site-directed mutagenesis (QuikChange)

What is the proper disposal of bacterial cultures and why is this important?

The proper disposal of bacterial cultures is through autoclave, which kills microorganisms by heating the material at a high temperature, 121 degrees Celsius, under a pressure of 103 kPa for atleast 15 min. Prevents contamination of the outside world

Why is agarose an ideal gel matrix for electrophoresis experiments?

The sugars of the agarose create a sieve to filter DNA fragments based on their size, and the greater the agarose concentration the smaller the pores within the gel. This allows you to control how the DNA will move through the gel.

You examine one of your control plates in Lab 7, pUC119 transformed cells on LBT, and notice colonies on the plate. What could be a likely explanation for this observation?

The tetracycline in the agar was not strong enough to kill sensitive colonies.

While loading the well of a gel with loading dye, you notice that the dye diffuses rapidly in the buffer that covers the gel, but doesn't go into the well. What could be the problem with your technique or the loading dye?

The tip of the pipette is not submerged completely into the well - Check that the tip is in the well before releasing the dye

What is the proper disposal protocol for pipette tips (plastic) that were used to set up a restriction digestion using purified DNA (no living cells used)?

The trash.

What contribution did Kary Mullis make to the development of PCR?

The use of "thermal cycling" and thermostable DNA polymerases

In the pACYC184 vector, what are the two means of selection for successful transformation? Why is a low copy number plasmid advantageous in situations when the investigator is expressing protein from the cloning vector?

The use of low copy plasmids reduces the total amount of expressed protein and can avoid production of inclusion bodies. LacZ' fragments almost always carry a MCS extension to the alpha-fragment DNA making these vectors extremely versatile.

Why might the actual tasting phenotype not match the phenotype predicted from this genetic test?

There are several bitter tasting receptor genes that encode different bitter tasting receptors, in addition to TAS2R38 This test only looked at one SNP in the three SNP haplotype for TAS2R38

Why might the actual tasting phenotype not match the phenotype predicted from this genetic test?

There are several bitter tasting receptor genes that encode different bitter tasting receptors, in addition to TAS2R38 This test only looked at one SNP in the three SNP haplotype for TAS2R38 Damage may have occurs to the taste receptors due to smoking or other damage that could impede the ability to taste.

How are bitter-tasting compounds recognized on the surface of the tongue? What types of food have compounds similar to PTC?

They are recognized by the sensory cells on the tastebud on the surface of the tongue. Vegetables

What are cells that are grown to mid-log phase used for and how long does it take for cells to reach mid-log phase in a standard culture?

They are used to make cells that are competent to take up DNA. It takes 3-4 hours for them to reach mid-log phase.

According to the module video, what was the immediate behavior of the fruit flies after being exposed to alcohol?

They become hyperactive

In addition to its large circular chromosome, many bacteria also carry DNA on what type of DNA? What controls the replication of this type of DNA in a bacterial cell?

They carry DNA in plasmids Plasmids replicate independently of the chromosome They are controlled by DNA sequences known as origins of replication (ori) Ori controls when plasmid replicates as well as how many copies of the plasmids exist in the cell

Why should an open bottle, tube or flask always be held at an angle when adding or removing liquid using sterile technique?

This minimizes the area open for contaminate to fall into the sterile container

How did you measure alcohol tolerance in the fruit fly mutants you studied in lab?

Timed how long flies took to show signs of intoxication and die relative to the non-mutants/wildtype

What is one equation used to calculate annealing T? Make sure you can apply this equation to a given primer sequence (see questions 13).

Tm = 4(G + C) + 2(A + T)

If you were given a sequence of a PCR primer, what simple equation would you use to calculate the annealing temperature that should be used in the 2nd step of each cycle of the PCR program?

Tm = 4(G + C) + 2(A + T) Ta = 4(G + C) + 2(A + T) - 5

What is the natural purpose of restriction endonucleases in bacteria?

To digest invading viral DNA

What were two reasons we needed to use specially treated cells in the transformation experiment in Lab 7 so that sufficiently transformed cells would result?

To obtain transformation from a ligation-cloning experiment you need cells with extremely high competency value. They also need to be able to take up the relaxed circle of the ligation- which is harder to transform.

The normal function of the MDR gene in the cell is to:

To pump out toxic chemicals in the cell

What is the purpose of the thermocycler?

To quickly and accurately heat and cool up to 96 0.2 ml tubes at once

If you wanted to separate fragments of DNA that were in the range of 50 base pairs long and differed from one another by only single nucleotides in length (e.g. 44 bps, 48 bps, 52 bps), what medium would you use to make a gel that can resolve these fragments and what percentage concentration would you use?

To separate DNA that was only a few base pairs in length I would choose to use an Acrylamide gel with a percent concentration closer to 20.0% or higher to increase resolution.

Mass of plasmid in cell suspension spread

Total mass of plasmid * fraction spread = Mass of Plasmid DNA Spread (ug)

Mass of plasmid used (ug): Concentration of plasmid: 0.005 ug/uL Plasmid solution added: 10 uL

Total mass of plasmid = volume * concentration (ug)

The method of DNA uptake by bacterial cells that uses a virus is called?

Transduction

What definition did Frederick Griffith give to the term: transformation, and how did he observe this phenomena while working with R-strains and S-strains of bacteria?

Transformation: a change in the genotype and phenotype by assimilation of a foreign substance. He observed this phenomenon by injecting a mixture of heat-killed S-strain (pathogenic) with R-strain into a host and the host died and upon autopsy found S-strain bacteria.

One specific combination of three SNPs (haplotype) correlates strongly with tasting ability for bitterness

True

Precision is defined as the degree of conformity between repeated measurements.

True

It would be more efficient to use two (and not one) restriction enzyme(s) to cut both the vector and the insert DNA, in preparation of cloning the insert DNA into the vector. True or False?

True.

QIAprep spin columns use a silica-gel membrane for selective absorption of the plasmid DNA in a high-salt buffer and elution in a low-salt buffer. True or False?

True.

Of the three types of restriction enzymes, which type is most useful in recombinant DNA experiments and why?

Type II They have identical recognition and cleavage sites and they cut DNA within the recognition sites.

Why are Type II restriction enzymes used in recombinant DNA experiments? In what important way do Type II restriction enzymes differ from both Type I and Type III?

Type II restriction enzymes are used in recombinant DNA experiments because they are simple and stable and they cut the DNA within the recognition site. Type II recognize specific 4, 5, 6 base pair sequences and cleave the DNA within these recognition sequences. Type I and III are much less specific, cutting tens to hundreds of base pairs away.

Why is double stranded DNA especially suited to size separation in electrophoresis?

Uniform structure and constant negative charge

What is one potential fire hazard in this laboratory course?

Using alcohol lamps while carrying out sterile technique.

How was a short region of the TAS2R38 gene amplified?

Using primers that flanked the region of the gene that contained one SNP

How was a short region of the TAS2R38 gene amplified?

Using primers that flanked the region of the gene that contained one SNP in a PCR reaction

In experiment Lab 7, why did you use 10x greater volume of ligated DNA in the transformation than control plasmid (pUC119 or pACYC184) when adding the DNA to the bacterial cells?

You expected the desired recombinant plasmid (pUC-tet) to be only one of many different plasmids made in the ligation mixture.

How do you make sure that all the liquid has been dispensed from the tip?

Verify by eye that the tip is empty

You observe that bacterial chromosomal DNA has contaminated your plasmid DNA. When could this problem have occurred?

Vortexing the neutralized lysate.

Who is credited for having their experimental approach transform biology into an experimental science?

Waston and Crick

Compare the transformation efficiencies expected for cells that have been specially prepared for transformation (as in Lab 7) versus for those cells you used in Lab 4, which used a simple and quick preparation method. Why do you think you recovered any transformants in Lab 4 if the cells had a lower expected transformation efficiency?

We are transforming cells with supercoiled DNA which is easier for the cells to take up and leads to a higher transformation efficiency. A lower expected transformation efficiency doesn't not mean that transformation cannot occur if the procedure is done carefully and the cells are given enough time to rest and create the desired gene products to compensate for the lowered efficiency.

Describe the mutation you will be reverting back to wildtype in this experiment. How will you recognize this event?

We will be reverting a nonsense stop mutation to a functional gene. It will be identified by plate analysis of the QC+/QC- plates yielding blue colonies

What is the normal function(s) of E. coli in the human colon (in both the absence and presence of O2)?

With Oxygen: Assist in waste processing Vitamin K production Nutrient absorption in the mammalian intestine Oxidize sugars into CO2 and water by oxidative phosphorylation Without Oxygen: Ferment sugars into lactic acid

Where is a Multiple Cloning Site usually positioned in a cloning vector and why?

Within the coding region of a marker gene for insertional inactivation screening.

What was one "new" device Morgan's lab used to induce new mutations in the genes of the fruit fly?

X-ray

Could you tell the difference between the plasmids from Colony 1 and Colony 2 without cutting them with Hind III and Eco R1, but rather just running them each out in separate lanes of a gel in the absence of digestion? Why?

Yes pUC-TET is larger, it would not run as far on the gel as pUC-CAP

Do you think it is necessary to use a Bunsen burner when you are using sterile glassware, pipettes and solutions?

Yes, use the burner to flame the edge of glassware when removing or adding liquid with a pipette

The tip on your micropipette has an air bubble in it after taking up a specified volume. What could be the problem?

You dialed in a volume in the pipette that was greater than what was in the tube.

While loading the well of a gel with loading dye, you notice that the dye goes into the well, but then goes right back up into the tip of the micropipette as you are pulling the micropipette out of the well. What could be the problem with your technique?

You did not keep the barrel depressed as you were pulling the pipette from the gel.

What is the equation used to calculate the annealing temperature of a set of primers?

[4(G+C) + 2(T+A)] - 5

Describe an experiment that you could conduct using PCR that would allow you to distinguish both Adh-F and Adh-fn23 from the wildtype Adh gene and from one another (assume the wildtype Adh gene is 1800 base pairs long). a. Describe the new elements of the experiment and how they will achieve the goal of the experiment: b. Expected results following PCR for each: - Wildtype DNA - Adh-F DNA - Adh-fn23 DNA

a. We could change the primers to flank the mutations and run the gel to check for more visible differences. The deletion and the insertion will become more obvious b. Wildtype and Adh-fn23 will be shorter than the Adh-F DNA. Adh-F DNA will run more substantially higher if run on a more concentrated gel, making the 37 base pair insertion more obvious. Adh-fn23 DNA will be shorter than the wildtype and the Adh-F DNA due to its deletion of the stop codon.

What two characteristics of Gregor Mendel's experimental design enabled him to recognize the two powerful mechanisms in genetics (Law of Segregation and Law of Independent Assortment)?

a. pure expression of characteristics b. Simple genetic mechanisms —> pure strains and controlled cross pollination

Biological Safety Level 1 refers to:

agents that can cause curable human disease

The term "in vivo" means:

carried out in a living organism

What genotype in the DH5alpha bacteria you used in this experiment enabled you to successfully introduce foreign DNA without that DNA being digested?

hsdR17(rK- mK+)

When you look at the genotype of DH5alpha (see table 3 in lab manual), what genes are deleted so that any DNA taken up by the cell will not be digested?

hsdR17(rK- mk+) prevents the up-taken DNA from being digested

What strains of bacteria are typically used in a lab at Biological Safety Level 1 and why?

most strains of non-pathogenic E. Coli because it is non pathogenic and poses no risk to human or environmental health

If the plasmids from Colony 1 and Colony 2 were purified and digested with Hind III and Eco R1 as done in Lab 8, how many restriction fragments and what sized restriction fragments would you expect for EACH plasmid?

pUC-CAP: 2 fragments (1.5 kb and 3.1 kb) pUC-TET: 2 fragments (3.7 kb and 3.1 kb)

You have recovered two LacZ-minus ampR colonies from your transformation in Lab 7 and have tested each of these colonies on an LB-tet plate. One of the colonies grew on the LB-tet plate (Colony 1) and one of the colonies did not grow on the LB-tet plate (Colony 2). What is the plasmid contained in Colony 1 and what is its total size? What is most likely the plasmid contained in Colony 2 and what is its total size? Be able to draw a simple plasmid map of each plasmid.

pUC-TET is colony 1 ~ 5.8 kb pUC-CAP is colony 2 ~ 4.6 kb

You have recovered two LacZ-minus AmpR colonies from your transformation in Lab 7 and they appear as white colonies on a LBAX plate. You test each colony on a LBT plate. Only one of these two colonies grew on the LBT plate (Colony 1) while the other colony did not grow on the LBT plate (Colony 2). Which is most likely the plasmid contained in Colony 2?

pUC-chloramphenicol(minus)

You have recovered two LacZ-minus AmpR colonies from your transformation in Lab 7 and they appear as white colonies on a LBAX plate. You test each colony on a LBT plate. Only one of these two colonies grew on the LBT plate (Colony 1) while the other colony did not grow on the LBT plate (Colony 2). What is the plasmid contained in Colony 1?

pUC-tet(resistant)

Which plasmid will be used as the cloning vector for this experiment?

pUC119

In the transformation of DH5alpha cells, what plasmid was used to make the positive control, negative control and experimental transformation?

pUC119: negative control with white colonies pACYC184: positive control with blue colonies pUC119/pACYC184 transformation: experimental with blue and white satellite colonies to show an insertion of a functional Lac Z gene

For E. coli cells, which form of DNA is easiest to transform (be taken up by the bacterial cell) (supercoiled, relaxed circles, or linear)?

supercoiled

7 tubes balanced in a 24 slot microcentrifuge: 1, 2, 7, 10, 13, 18, 19

yes