MCB 251

What are the benefits of taking the newly assembled plasmid (vector+insert) and putting it into a bacterial cell?

- You can quickly screen the bacterial cells for antibiotic resistance (and therefore the presence of your successfully ligated plasmid). - Allows you to easily replicate the your newly constructed plasmid. Then you can produce many copies of the plasmid for use in subsequent reactions - making it a renewable resource.

Describe the molecular mechanisms of PCR, focusing on the importance of each temperature condition, the significance of the enzyme used, and the purpose of the final extension.

-95°C to denature double stranded DNA -Tm-5°C (typically 50-60°C) for primers annealing to complementary sequence on the template DNA --The melting temperature depends on the G/C content of the primer sequence -72°C for Taq DNA polymerase to add new nucleotides to the growing strand --Taq was isolated from thermophilic bacteria -Cycle repeated ~30x for amplification -Final extension is to allow polymerase to extend the underrun strands generated due to the stochastic nature of polymerase

You experiment requires you to add 7 μl of a reagent into a microcentrifuge tube. On the display panel of the chosen pipette, how will you se the volume? Indicate the numbers from top to bottom.

0 7 0

What is the volume if a P200 micropipette is set to the following: 0 5 2

0.052ml

Based on the transformation results, you suspect that your insert encoded the kan gene, but being a good scientist, you want to verify this finding through another method. Using the colonies growing on the LA + kanamycin plate as your starting point, describe 2 additional methods of identification. (Hint: Think back to the techniques we've covered in class so far.)

1) Inoculate the colony in LA broth containing either chloramphenicol or kanamycin. Growth is only expected in the kanamycin-containing broth. 2) Either isolate plasmid from the colony or boil colony and PCR amplify the insert using the primer set that recognizes both inserts. The size difference on gel electrophoresis (1.5 vs 1 kb) will indicate the insert. Given the transformation results, you'd expect a band around 1.5 kb. 3) Inoculate the colony as in #1, isolate the plasmid, and perform restriction digestion with either BamHI (and look for the size of the insert at ~1.5 kb), PstI

Assuming that you start with one template DNA molecule, how many total DNA molecules would you have after 30 cycles? 1.07 e9 60 1073 6.48 e5

1.07e9

5 μl of the purified vector plasmid with a concentration of 200 ng/μl is used for a digest reaction where the total volume is 20 μl. After digestion, you clean out the restriction enzyme from the entire digestion product using the in silica QIAGEN column. If you use 50 μl of Buffer EB to elute the DNA off of the column, what is the new concentration of the digested and cleaned vector plasmid? -80 μg/μl -20 μg/μl -80 ng/μl -20 ng/μl

20 ng/μl

Assuming that you start with one template DNA molecule, how many target DNA molecules would you have after 8 cycles? 256 240 16 not enough information to answer this question

240

Taq DNA polymerase adds nucleotides to the __ end of the growing DNA strand. 5' 3'

3'

You clone in the kanR gene (confers resistance to kanamycin) at the EcoRI site in pBLU, perform transformation into E. coli DH5α cells, and plate the transformants onto the following plates: LA, LA + ampicillin, LA + ampicillin + X-gal, LA + kanamycin. d. What color(s) are the colonies growing on the LA + ampicillin + X-gal plate? - All colorless/white colonies because the lacZ gene encoded in pBLU was disrupted by the kanR gene - A mixture of blue and colorless/white colonies because the blue colonies indicate transformed cells and the colorless/white colonies indicate untransformed cells - All blue colonies because the lacZ gene encoded in pBLU remained intact - A mixture of blue and white colonies because the ligation product has a mixture of self-ligated pBLU and those that ligated with the kanR gene

A mixture of blue and white colonies because the ligation product has a mixture of self-ligated pBLU and those that ligated with the kanR gene

When graphing the distance traveled vs the size of DNA fragment, what is a benefit of drawing a best-fit line verses connecting the data points?

Because the best-fit line is generated by the DNA standard bands, it can be used to estimate the size of the DNA fragments based on the distance travelled.

You cloned in your insert DNA into pBLU at the BamHI site and plated the transformants on the LA + ampicillin + X-gal plate to perform the blue/white screen. After an overnight incubation at 37ºC, you observe a mixture of blue and white colonies. Indicate the following: -The molecular difference between the two populations/what is the genetic make up of a white colony versus a blue colony

Blue: The lacZ gene is intact, indicating that the insert did not disrupt the gene.White: The lacZ gene is disrupted by the insert.

After transforming the plasmid DNA into E. coli DH5α, you skip the recovery period because you think this is a waste of your time and plate the transformants onto LA with an appropriate antibiotic. Why is this a bad idea? -More time is required to incorporate the plasmid DNA through the cell membrane. -Cells need time to express the antibiotic resistance gene. -Larger DNA plasmids take longer to undergo chemical transformation. -It's fine. E. coli is hardy so they will grow just fine.

Cells need time to express the antibiotic resistance gene.

True or false? -Chemical transformation and electroporation are examples of artificial transformation. -E. coli DH5α is able to take up foreign DNA through natural transformation. -Calcium shock is an example of electroporation. -Natural transformation only work by electroporation.

Chemical transformation and electroporation are examples of artificial transformation.

Which of the following statements is not a definitive characteristic of a plasmid? -Smaller than the host chromosomal DNA -Extrachromosomal DNA -The loss of a plasmid does not impact cell viability under conditions with no added selection -Circular

Circular

Describe (generally) what happens to the membrane of competent cells when heat-shocked during the transformation protocol.

DNA is attracted to calcium on the membrane. Sudden heat exposure disrupts the membrane, allowing DNA to slip inside. Ice returns the membrane to a more stable state.

Which of the following statements best describes a DNA ladder? -DNA ladder functions to weigh down your DNA sample in the agarose gel -DNA ladder contains a mixture of DNA fragments of known sizes that can be used a a point of comparison for your DNA sample(s). -DNA ladder acts as a scaffold, allowing DNA to travel down the gel -Incorrect DNA ladder ensures your DNA fragments to run through the agarose gel efficiently

DNA ladder contains a mixture of DNA fragments of known sizes that can be used a a point of comparison for your DNA sample(s).

You realize your mistake, and you are ready to tackle the second round. You program the PCR thermocycler with the following conditions: 1. 5 min 94°C2. 30 sec 94°C3. 30 sec 72°C4. 30 sec 50°C5. Repeat Steps 2~4 for 30 cycles6. 5 min 72°C7. Hold at 4°C You run DNA the PCR polymerase product on a extension gel, but cannot yet again, you do not see a band. Why?

DNA polymerase extension cannot occur prior to primer annealing

Which of the following statements about gel electrophoresis is true? -Larger DNA fragments will migrate at a faster rate than smaller DNA fragments -Agarose is a mixture of agar and agaropectin that forms a repeating and crosslinked disaccharade -DNA will migrate from anode to cathode on agarose gel -DNA will migrate towards the positively charged electrode

DNA will migrate towards the positively charged electrode

Which of the following is the best way to avoid concatenation? -Digest the cloning vector with a "blunt end"-generating restriction enzyme prior to ligation -Plate transformants onto LA with ampicillin -Shorter ligation time -Digest the cloning vector and the insert DNA with two different "sticky end"- generating restriction enzymes prior to ligation

Digest the cloning vector and the insert DNA with two different "sticky end"- generating restriction enzymes prior to ligation

You clone in the kanR gene (confers resistance to kanamycin) at the EcoRI site in pBLU, perform transformation into E. coli DH5α cells, and plate the transformants onto the following plates: LA, LA + ampicillin, LA + ampicillin + X-gal, LA + kanamycin. a. What is a possible explanation if no growth is observed on the LA plate? -The kanR gene did not ligate with pBLU -The water bath for the heat-shock procedure was set to 100ºC -E. coli DH5α cells were not viable and the water bath for the heat-shock procedure was set to 100ºC -The kanR gene did not ligate with pBLU and pBLU was not digested by EcoRI fully -pBLU was not digested by EcoRI fully and the water bath for the heat-shock procedure was set to 100ºC

E. coli DH5α cells were not viable and the water bath for the heat-shock procedure was set to 100ºC

Which of the following is a drawback of PCR? -Amplification of a targeted region of DNA -Simplicity in experimental design -False-positives due to high sensitivity -Cutting DNA using restriction enzymes

False-positives due to high sensitivity

When setting up restriction digestion reactions, which of the following is not a reason to have a negative control (i.e. without restriction enzymes)? -To check for DNase contamination -For verifying the enzymatic activity -That the isolated plasmid was not already digested -A point of comparison for undigested vs digested DNA

For verifying the enzymatic activity

When designing PCR primers, which primer (forward vs reverse) anneal to which DNA strand (coding vs template)?

Forward primer to template strand, reverse primer to coding strand

You are given the coding strand sequence of the region that you are interested in PCR amplifying. Design forward and reverse primers using the sequences highlighted in yellow. Your primer sequences should be written in a 5' → 3' direction. refer to lon capa

Forward: GTCGAGATCTTACGATAACReverse: GACTTCGACATGAACCATG

You don't know which insert you have, and the inserts are different sizes, meaning the amount needed for a 1:3 ratio is different. What do you do? Why? (assume there are enough materials for more than one reaction per person).

Free answer, they could either set up two reactions, one at each appropriate 1:3 concentration, or they could just use the higher number... I want to see their reasoning.

Which of the following statements about the 3:1 molar ratio (insert to vector plasmid) for ligation is false? -Increases the chance of the insert filling the gap within the vector plasmid -Reduces the risk of concatenation -Increasing the digestion efficiency -Minimizes self-ligation of the vector plasmid

Increasing the digestion efficiency

You clone in the kanR gene (confers resistance to kanamycin) at the EcoRI site in pBLU, perform transformation into E. coli DH5α cells, and plate the transformants onto the following plates: LA, LA + ampicillin, LA + ampicillin + X-gal, LA + kanamycin. c. What is the importance of having ampicillin in the X-gal plate in order to perform a blue/white screen?

Inhibits the growth of untransformed cells, which would appear as colorless. Therefore, the colorless colonies indicate the cells transformed with the modified plasmid. Also, you can observe individual colonies to differentiate the different populations

You are working as an undergraduate researcher in a microbiology lab. You and your graduate student research mentor identify a bacterial toxin (you name it Mcb251) that is released by the bacteria during infection. You come up with the following hypothesis: Mcb251 introduces double strand breaks to DNA Design an experiment including a positive control and a negative control to test your hypothesis. (Keep your answer concise)

Introduce Mcb251 to DNA and load on agarose gel Positive control: treating DNA with something that can cut DNA (e.g. restriction enzymes, DNAase) Negative control: no DNA break

What is the main purpose of isopropanol and ethanol in Bugger PB and Buffer PE, respectively? -Renaturation of DNA -Keeping DNA insoluble -Denaturation of proteins bound to DNA -Eluting DNA off the column

Keeping DNA insoluble

After ligating the insert into the vector, you perform transformation and plate the transformants onto the following plates: LA, LA + ampicillin, LA + chloramphenicol, LA + kanamycin. Describe the molecular reasonings for why you observe or do not observe growth on each plate, and how the results help you identify the insert.

LA: all cells plated are expected to grow. LA + ampicillin: all transformants expected to grow (regardless of if the plasmid contains the insert or not). LA + chloramphenicol: only the transformants that internalized the plasmid with the cat gene will grow. LA + kanamycin: only the transformants that internalized the plasmid with the kan gene will grow. The growth on chloramphenicol or kanamycin will indicate which insert you have.

You are tasked with PCR amplifying a certain gene of interest. a. To set up the reaction, you add appropriate volumes of diH2O, template DNA, Taq DNA polymerase, buffer, forward/upstream primer, and reverse/downstream primer into a tube and run the sample in a PCR thermocycler. After completion, you run the PCR product on a gel, but you do not see a band. Why?

Lacking dNTP

You add 1 ml of bacterial culture to 9 ml of saline (Tube A). Then you take 1 ml of Tube A and add to 99 ml of saline (Tube B). Calculate the local and global dilution factors of Tube B.

Local DF: 102Global DF: 103

How does an agarose gel function to separate DNA fragments? -a solid growth medium -molecular sieve -denatures DNA -amplifies DNA

Molecular sieve

After running your PCR to determine your unknown insert you run a gel to visualize your resulting product. Explain your finding for each of the following results: multiple bands, all different lengths ranging from 0.5 - 4.0 kb

Multiple segments of DNA were amplified. Possibly due to non-specific primers.

UiuC is a protein toxin that secreted by a particular bacterial pathogen that targets the mitochondria upon entering a mammalian host cell. Based on a literature search, you decide to test the hypothesis that UiuC damages mitochondria by disrupting the electron transport chain. Which of the following options contains the correct conditions for negative control, positive control, and experimental group? (Hint: CCCP is a small molecule that disrupts mitochondrial membrane potential in cells. Saline has no effect on cells.) Negative control = cells incubated with CCCP; Positive control = cells incubated with CCCP; Experimental group = cells incubated with UiuC Negative control = cells incubated with UiuC; Positive control = cells incubated with CCCP; Experimental group = cells incubated with saline Negative control = cells incubated with saline; Positive control = cells incubated with CCCP; Experimental group = cells incubated with UiuC Negative control = cells incubated with saline; Positive control = cells incubated with UiuC; Experimental group = cells incubated with CCCP

Negative control = cells incubated with saline; Positive control = cells incubated with CCCP; Experimental group = cells incubated with UiuC

You cloned in your insert DNA into pBLU at the BamHI site and plated the transformants on the LA + ampicillin + X-gal plate to perform the blue/white screen. After an overnight incubation at 37ºC, you observe a mixture of blue and white colonies. Indicate the following: -Whether the results allow the insert identity to be determined.

Neither colors will indicate the identity of the insert.

You and your graduate student research mentor are isolating plasmid pMCB251 from E. coli. After centrifuging the E. coli culture, you discard the supernatant and resuspend the pellet with 250 μl of Buffer P1. You then add 350 μl of Buffer N3. Will your plasmid isolation be successful? Why or why not? -Yes, because adding Buffer N3 will precipitate cell debris and denatured DNA and proteins. -No, since RNase from Buffer P1 must react with NaOH in Buffer P2 for its maximal activity. -Yes, since you will subsequently add Buffer P2. -No, because NaOH and SDS in Buffer P2 are required to disrupt the cell membrane and denature DNA and proteins.

No, because NaOH and SDS in Buffer P2 are required to disrupt the cell membrane and denature DNA and proteins.

Will the orientation of an insert DNA that contains a gene and its promoter affect its gene expression? Why? -No, because transcription will occur if a promoter is placed upstream of the gene. -Yes, because transcription requires a promoter upstream of the gene. -Yes, because translation requires the gene to be in a forward orientation. -No, because the lacZα gene will be disrupted by the insert DNA.

No, because transcription will occur if a promoter is placed upstream of the gene.

The forward and reverse primers for PCR amplification have the following sequences: Forward: 5'-CTATGAGTTACTCACATG-3', Reverse: 5'- GAAAGCATCCAGGCCCGG-3'. Based on the manually calculated melting temperatures, would you recommend using these primers together? Why or why not?

No. The difference in the primer melting temperatures is >5ºC.

After running the digestion product on an agarose gel, you unexpectedly see one band that corresponds to the linear full-length size of the plasmid. What technical error might have caused this issue? -One of the enzymes lost enzymatic activity -Introduced EcoRV instead of EcoRI -Added more Peil than EcoRI -Added too much Buffer PE

One of the enzymes lost enzymatic activity

You experiment requires you to add 7 μl of a reagent into a microcentrifuge tube. Which pipette would you use? P5000 micropipette P200 micropipette P1000 micropipette P20 micropipette

P20

During the digest clean up what buffers are used and what is the purpose of each one?

PB - Contains Guanidine hydrochloride to bind the DNA to the silica membrane while removing restriction enzymes PE - Wash Buffer containing Tris Buffer and 75% ethanol to keep the DNA in solution while washing away salts and guanidine Hydrochloride EB - Elution Buffer using tris to dissolve the DNA and elute it from the silica

After running your PCR to determine your unknown insert you run a gel to visualize your resulting product. Explain your finding for each of the following results: Nothing (Explain possible reasons)

PCR did not amplify

Pertaining to the second mode of analysis for the unknown insert identification project, match the following statements to PCR, restriction digestion, or both. -Amplify a specific region of DNA -Using a different vector plasmid yields a different outcome -Requires controlled temperature changes using a thermocycler -Gel electrophoresis is required for analysis -Requires the knowledge of predicted DNA fragment size for analysis -Isolating purified plasmid is necessary for analysis

PCR: -Amplify a specific region of DNA -Requires controlled temperature changes using a thermocycler restriction digestion: -Using a different vector plasmid yields a different outcome -Isolating purified plasmid is necessary for analysis both: -Gel electrophoresis is required for analysis -Requires the knowledge of predicted DNA fragment size for analysis

You want to check if a plasmid in E. coli contains a specific gene of interest. If you are given a liquid culture of this strain, what should you do? -Plasmid isolation, transformation into E. coli DH5a, digestion with BamHI - Plasmid isolation, ligation, PCR, gel electrophoresis -Plasmid isolation, PCR, transformation -Plasmid isolation, PCR, gel electrophoresis

Plasmid isolation, PCR, gel electrophoresis

You use PCR as your second approach to identify your unknown insert. What is your positive control, negative control, and experimental group?

Positive control = amplify from the insertNegative control = amplify from your initial, unligated plasmidExperimental group = amplify from the ligated vector + insert

DNA polymerases have a ring-like structure that clamp on DNA and can dissociate from the DNA at some frequency. Enzymes that are more likely to remain onto the DNA longer than others have a higher: Progeny Plasmid Protectivity Processivity

Processivity

Each colony on a solid growth medium is a(n): -Identification of unique species -Progeny from a single cell -Contaminant -Enumeration of cell concentration

Progeny from a single cell

When graphing the distance traveled vs the size of the DNA fragment, what type of graphing technique is applicable and why? -Linear graph because of its accuracy -Linear graph because it allows for the expansion of smaller numbers so they can be differentiated on the graph -Semi-log graph because it allows for the compression of large number to allow for all data to be presented in a smaller graph -Semi-log graph because it allows for regularly linear data to be presented in a curved matter.

Semi-log graph because it allows for the compression of large number to allow for all data to be presented in a smaller graph

You digest a plasmid with a restriction enzyme that recognizes a single site on the plasmid. When you perform gel electrophoresis on the digestion product, you quickly realize that there are two bands; one at the expected size and one near the well. Which of the following best explains the outcome? -The some plasmids ligated together -DNA was trapped in the agarose gel -The presence of the chromosomal DNA -Some plasmids were not digested

Some plasmids were not digested

What is one advantage and one disadvantage for using sticky-end generating restriction enzymes for cloning? How about for blunt-end generating restriction enzymes?

Sticky end: The overhang generated by the staggered cut allows for higher ligation efficiency, but the restriction recognition sequence may not be available in either the vector plasmid or the inserting DNA. Blunt end: No sequence specificity is necessary for ligation, but the ligation efficiency is lower than when a sticky-end generating restriction enzyme is used.

True or false -DNA will travel across an agarose gel according to its mass -the current runs from cathode to anode -cathode is the positively-charged electrode -the casting tray is required for DNA gel electrophoresis -DNA loading dye contains glycerol to weigh down DNA in wells

TRUE the casting tray is required for DNA gel electrophoresis DNA loading dye contains glycerol to weigh down DNA in wells

Your graduate student research mentor tasked you with setting up a ligation reaction. The protocol indicated to mix the insert DNA and the cloning vector at 3: 1 molar ratio. However, you were distracted (your lab mate started talking to you) and mixed the insert DNA to the cloning vector at 1 :3 molar ratio instead. How would this affect the outcome? -The ligation efficiency will remain the same. -The ligation efficiency will be lower because decreasing the amount of insert DNA will lower the chances of inserting the insert DNA between the opening on the cloning vector. -The ligation efficiency will be higher because more cloning vector is available to take up the insert DNA. -Needs for more information.

The ligation efficiency will be lower because decreasing the amount of insert DNA will lower the chances of inserting the insert DNA between the opening on the cloning vector

You have identified a gene expressed by a certain bacterial pathogen. You decide to clone this gene into a plasmid vector. Which of the following statements is a factor to consider when choosing the vector between a high copy number plasmid and a low copy number plasmid? -The plasmid copy number does not matter for cloning -The toxicity of the gene product to the host cell -Containing components necessary for conjugation (oriT or mob) -The size of the plasmid

The toxicity of the gene product to the host cell

What is the typical annealing temperature range used for PCR? What is important about this range?

The typical annealing temperature ranges from 40ºC-60ºC. This is because the Tm must be determined for each primer, as they are designed to fit the DNA sequence that is to be amplified and is not always the same sequence. If the temperature is too high the primers would not anneal because they would be denatured.

What is the purpose of using the same restriction enzyme for digesting the insert DNA and the vector plasmid? Assume that the restriction enzyme produces sticky ends. -To orient the insert towards a uniformed direction -To avoid self-ligation of the vector -To avoid concatenation of the insert -To generate compatible ends for ligation

To generate compatible ends for ligation

Transformation b. What is the importance of the recovery period after performing heat-shock? -The recovery period is optional -Cells express tools for surviving in the presence of antibiotics -To cool down the cells after heat-shock -To increase cell viability by allowing cells to recover from a stressful event and to cool down the cells after heat-shock event -To increase cell viability by allowing cells to recover from a stressful event and cells express tools for surviving in the presence of antibiotics

To increase cell viability by allowing cells to recover from a stressful event and cells express tools for surviving in the presence of antibiotics

What is the purpose of a three way streak? -To isolate a single colony -To quantify the number of bacteria present -To spread the bacteria around -To compare bacterial growth

To isolate a single colony

You clone in the kanR gene (confers resistance to kanamycin) at the EcoRI site in pBLU, perform transformation into E. coli DH5α cells, and plate the transformants onto the following plates: LA, LA + ampicillin, LA + ampicillin + X-gal, LA + kanamycin. b. What is the purpose of plating on the LA + ampicillin plate?

Transformation efficiency

True or False? -The ends produced by the endomuclease can be rejoined by a ligase, which closes the break in the hydrogen bonds formed by the complementary DNA nucleotides -Ampicillin is a gene that encodes for ampicillin resistance -During DNA ligation, DNa fragments with compatible sticky ends have a higher ligation efficiency than DNA fragments with blunt ends -Restriction digestion is required for PCR amplifying DNA -PCR is a technique used to amplify a region of DNA defined by the forward/upstream primer and the reverse/downstream primer

True: -During DNA ligation, DNA fragments with compatible sticky ends have a higher ligation efficiency than DNA fragments with blunt ends. -Restriction recognition sites typically have a palindrome structure. -PCR is a technique used to amplify a region of DNA defined by the forward/upstream primer and the reverse/downstream primer. False: -The ends produced by the endomuclease can be rejoined by a ligase, which closes the break in the hydrogen bonds formed by the complementary DNA nucleotides -Ampicillin is a gene that encodes for ampicillin resistance -Restriction digestion is required for PCR amplifying DNA.

Why do undigested and digested plasmids run differently on an agarose gel, even if they both have the same number of base pairs? -Undigested plasmid DNA is linear while digested plasmid DNA is open circle -Undigested plasmid DNA is linear while digested plasmid DNA is supercoiled -Undigested plasmid DNA is supercoiled while digested plasmid is open circle -Undigested plasmid DNA is supercoiled while digested plasmid DNA is linear

Undigested plasmid DNA is supercoiled while digested plasmid DNA is linear

Which of the following treatments can reduce the frequency of self-ligation in the vector plasmid? -Remove restriction enzymes from the vector and insert before ligation - Use a high insert to vector molar ratio in ligation -Use a high insert to vector molar ratio in ligation and digest the vector and use two different restriction enzymes to digest both vector and insert -Use two different restriction enzymes to digest both vector and insert and remove restriction enzymes from the vector and insert before ligation - Use a high insert to vector molar ratio in ligation and remove restriction enzymes from the vector and insert before ligation

Use a high insert to vector molar ratio in ligation and digest the vector and use two different restriction enzymes to digest both vector and insert

What is self-ligation? -When the vector plasmid ligate to itself instead to the insert DNA -When multiple insert DNAs are ligated together within the vector plasmid -When the insert DNA is digested using a "blunt end"-generating restriction enzyme -When ligation is complete and ready for transformation

When the vector plasmid ligate to itself instead to the insert DNA

Why is it necessary to find the new vector plasmid concentration after undergoing digestion and the clean up?

When the volume changes, the concentration changes too.

Let's say you transformed E. coli DH5-alpha cells with or without a plasmid encoding ampR (ampicillin resistance gene). You then plate these cells on LA and LA + ampicillin plates, and incubate them overnight at 37°C. Explain the growth you would observe on each of the plates. (You should have 4 plates total)

With plasmid on LA: Lawn, all cells are expected to growWith plasmid on LA + ampicillin: only the transformed cells will growWithout plasmid on LA: Lawn, all cells are expected to growWithout plasmid on LA + ampicillin: no growth

You used pBLU as the vector for the unknown insert project. After transformation, you decide to perform a blue/white test, so you plate the transformants on LB + ampicillin with X-gal. Why is ampicillin required in this plate?

X-gal is not an antibiotic, so it does not select for cells that picked up a plasmid.

In which of the situations below would you have a definitively higher chance of identifying your insert using PCR, rather than restriction digest? (Assume you have purified the insert-containing plasmid from chloramphenicol or kanamycin resistant E. coli.) -Your purification goes particularly well and you end up with a very high concentration of plasmid. -Your tube gets dropped, spilling all but ~1 μL of purified plasmid solution in a small droplet that you can still capture with your pipette. -The 37 degree incubator is broken, so you can't put your reaction in there for an hour. -Your plasmid vector is pAMP.

Your tube gets dropped, spilling all but ~1 μL of purified plasmid solution in a small droplet that you can still capture with your pipette.

If growth is observed on LA and LA + ampicillin plates, but not on either LA + chloramphenicol or LA + kanamycin, what step(s) might have failed? How could you verify this? Assume some ligation reaction volume was left over.

a. Digestion may have failed, or insert may not have been added or added at a non-ideal concentration, leading to mostly re-closing of the vector without the insert. b. Run ligations on a gel and/or perform PCR with insert primers

If growth is observed on LA plates only, which step(s) most likely failed? How could you verify this? Assume some ligation reaction volume was left over.

a.Transformation, or any step that transfers liquid from one container to another, or incorrect buffer order in digest clean-up. b. If any ligation volume is left, run that on a gel or perform PCR to verify that the vector exists. c. Could have run gels between each step to verify presence/concentration.

Which of the following can cause inaccuracy when transferring solution using a micropipette? - Keeping the pipette at 90 degrees -Air bubbles - Slowly drawing up the solution - Immersing the pipette slightly below the surface of the solution

air bubbles

You are preparing a bottle of LB brother for growing a culture of E. coli. Which of the following methods is the best for sterilization? Ionizing radiation Sterile filtration Autoclave Add 10% bleach

autoclave

After running your PCR to determine your unknown insert you run a gel to visualize your resulting product. Explain your finding for each of the following results: One band near 1.0 kb

cat insert

Restriction enzymes are used by bacteria to __________ and utilized by scientists to __________. -nonspecifically degrade DNA; specifically degrade DNA -synthesize DNA; synthesize RNA -replicate; amplify a region of DNA -degrade foreign DNA; clone a DNA fragment into a plasmid DNA

degrade foreign DNA; clone a DNA fragment into a plasmid DNA

Which of the following statements is the main reason for using agar as the solid base in growing microorgamisms? -relatively low melting temperatures -metabolized by many bacterial species -hysteresis -separates DNA fragments by size

hysterisis

You want to PCR amplify a certain gene and know that a colleague has worked with it before. You ask if they have some primers you can use and they say yes, and hand you two tubes with the sequence printed on the side. "They're kind of low-efficiency, but they work at about 56°C!" your colleague says. Sure enough, you use them in a PCR with a 56°C annealing temperature, run the resulting solution on a gel, and see a somewhat faint band at the expected size. You also observe a brighter band towards the bottom of the gel, further down than even the smallest band in your standard (100 bp). You look at the primer sequence (below) and notice some non-ideal things about their design. List at least 2 deviations from the primer design guidelines you were provided, and how those may have impacted your results.F: 5'-AAGATAGGGGCATATAGCT-3'R: 5'-AGGCCCCCCTATCTTGAGT-3'

i. Lack of G/C at either end: leads to reduced amplification efficiency ii. Significant homology shared between primers: primer-dimer formation, reduced amplification efficiency iii. Significant hairpin present in reverse primer: reduced amplification efficiency. iv. Palindromic 3' end of forward primer: reduced amplification efficiency and primer-dimer formation.

After running your PCR to determine your unknown insert you run a gel to visualize your resulting product. Explain your finding for each of the following results: a. One band near 1.4 kb

kan insert

Transformation a. Which of the following techniques are not used to introduce foreign plasmid DNA into bacterial cells that do not spontaneously take up DNA? -calcium shock -natural transformation -artificial transformation -electroporation

natural transformation

Which of the following is not a tool used to transfer bacteria? -pipet -inoculating loop -non-sterile microcentrifuge tube -micropipette

non-sterile microcentrifuge tube

Which of the statements is false? -the experimental design is flawed if you observe a "positive" phenotype in the negative controls -positive control is necessary to test the validity of the experiment -only positive and negative control are necessary to test the hypothesis -negative control is a condition to test for confounding variables

only positive and negative controls are necessary to test the hypothesis

What is not is a characteristic of cloning vectors? -Usually contain multiple cloning sites -Carry antibiotic resistance gene(s) that can be used for selection -Single-stranded DNA -Typically circular

single-stranded DNA

What is the purpose of spread plates? -To compare bacterial growth -To isolate a single colony -To quantify the number of bacteria present -To spread the bacteria around

to quantify the number of bacteria present

Which of the following methods is not considered a sterile technique? bleach washing with soap and water autoclaving fire (flaming loop)

washing with soap and water