Postlab Final

Identify the article title below that would be considered pseudoscience.

"The Crop Circle Mystery: A Closer Look" - Live Science

Identify the technique that each statement corresponds to: Restriction Digestion, PCR, or Both

- Both: Samples can be run on a gel to visualize bands - Restriction Digestion: Restriction enzymes are used to digest phosphodiester bonds - PCR: Primer dimers can develop and may be visible when samples are run on the gel - Both: Products on a gel (bands) can be analyzed with a semi-log graph to obtain sizes of bands - PCR: Uses cycles of melting DNA, annealing of primers, and extension with DNA polymerase - Restriction Digestion: Digested DNA will appear as the product on the gel - Restriction Digestion: Blunt ends or sticky ends are formed during this reaction - PCR: Target strands are formed during cycling and will appear as the products visualized on a gel

Identify each statement as TRUE or FALSE.

- False: Primers bind to sticky ends that are created during a restriction digestion - True: Both the plasmid and the insert are digested with the same restriction enzymes when used for cloning - False: DNA polymerase (such as Taq polymerase) is used to digest phosphodiester bonds in a restriction digestion reaction - True: Restriction enzymes cut DNA at a restriction (recognition) site - False: Primers are used in restriction digestion reactions - False: Products from a PCR reaction have sticky ends and can be used immediately for ligation - True: Primers anneal to single-stranded DNA created during the DNA denaturation (melting) step - False: Temperature cycling is used during a restriction digestion reaction to cut phosphodiester bonds

Match the definition with the term.

- Flanking Regions: Base pair sequence around a segment of DNA that is not variable - Short Tandem Repeats: DNA variations that are units of bases that repeat multiple times - Loci: The DNA sequence at a certain location - Allele: Variant or alternative form of a gene; STRs can have varying repeat units in a gene - Heterozygous: Different number of repeats in the STR of both alleles of a gene - Homozygous: Same number of repeats in in the STR of both alleles of a gene

Choose if either statement belongs to restriction digestion, polymerase chain reaction (PCR), or both.

- PCR: Has cycling of three temperatures for DNA melting, primer annealing, and extension - Both: Analyzed by running products on a gel - PCR: Used to amplify regions of DNA - PCR: Uses primers - PCR: Uses Taq polymerase for the extension step - Restriction Digestion: Used to open a plasmid to be used in cloning - Restriction Digestion: Used to cut plasmids into pieces - Restriction Digestion: Uses restriction enzymes for the digestion

Match the correct term with the definition. Refer to the lab manual and the PowerPoint.

- Sticky Ends: DNA that has overhangs after digestion with restriction enzymes - Restriction: Double-stranded cutting of DNA - Ligation: Joining of two DNA strands - Cloning Vector: Plasmid used in cloning - DNA Ligase: Used to repair nicks and breaks in phosphodiester bonds - Blunt Ends: DNA that does not have overhangs after digestion with restriction enzymes

Mark TRUE for the correct possibility for how an insert can be ligated into a plasmid. Mark FALSE for incorrect possibilities.

- True: Concatentation (multiple inserts are ligated together and with the vector) -True: In the reverse orientation compared to the lacZα gene -False: In the forward orientation compared to the lacZδ gene -True: In the forward orientation compared to the lacZα gene -True: Not ligated (self-ligation of the plasmid) -False: In the reverse orientation compared to the lacZδ gene

Mark "YES" or "NO" for if this primer set follows the guidelines. Forward: 5'- CCT TCC TTA CCT TAA GGA AGG - 3' 10/21 40+22Reverse: 5'- CCT TAC CCA CCC TAA CCC ACC - 3' 13/21 52+16

- Yes: No more than two G/C's at the 5' and 3' ends for each primer - No: G/C content between 40% and 60% for each primer - No: Tm between 50-60°C for each primer - No: No self-complementarity for each primer - No: There are no complementary sites in this set - Yes: Each primer is between 18 and 24 base pairs long - No: There are no runs of G/C's longer than three base pairs - No: Differences in melting temperature does not exceed 5°C

Identify which guidelines the primer pair follow. Select "Yes" if the primer pair follows the guideline, and "No" if the primer pair does not follow the guideline. Forward: 5'- ATC CGG GGG CGC ATT TTA TGG -3' 48+18=66Reverse: 5'- TTC CTA TAA GCC TAA GCT TGC -3' 36+24=60

- Yes: One or two G/C at the 3' end - No: Melting temperature between 50-60ºC for both primers - Yes: Falls within the range of 18-24 bp for both primers - No: G/C nucleotides distributed equally along the primer sequence for both primers - Yes: G/C content between 40-60% for both primers - No: Primer pair has a Tm within 5ºC of each other

In no more than two sentences, explain the two ways that restriction enzymes are used during cloning.

1) Restriction enzymes are used to digest the plasmid and insert to give sticky ends before the ligation reaction 2) Restriction enzymes are used to digest the new plasmid to give a unique digestion pattern to analyze if the insert was ligated and to tell the size of the insert if the size is unknown.

Put the steps for a transformation in the correct order (#1-9).

1. Add uncut purified plasmid to DH5α cells 2. Incubate on ice for 30 minutes 3. Heat shock plasmid in 42°C water bath for 90 seconds 4. Place tube on ice for 2 minutes 5. Add 800μL SOC broth to microfuge tube 6. Allow tubes to shake for 1 hour at 37°C 7. Add 100μL of DH5α cell/plasmid/broth mixture to a LB/ampicillin plate 8. Use the spread plate technique to spread sample on LB/ampicillin plate 9. Incubate plates at 37°C

Place the steps in order to identify an unknown insert.

1. Digest a pBR322 vector and unknown insert (kan or cat) with the same restriction enzyme 2. Stop the digestion with the restriction enzyme using a plasmid clean-up kit 3. Use T4 DNA ligase with an insert to vector ratio of 3:1 for the ligation reaction 4. Conduct a transformation of the pBR322 with the unknown insert and plate onto antibiotic resistance plates (kanamycin or chloramphenicol) and grow at 37°C 5. Pick a colony from the antibiotic resistance plate with growth (this will give the first clues as to which insert you have) and put into LB with appropriate antibiotic and incubate at 37°C for growth 6. Isolate the pBR322 plasmid with the unknown insert using the Qiagen kit 7. Digest the pBR322 plasmid with unknown insert with restriction enzymes to make a unique digestion pattern or use PCR with primers targeting the insert to analyze the insert 8. Run the products from the restriction digestion reaction or the PCR on a gel to analyze which insert you have

Put the steps in correct order for cloning the pBAMI plasmid with the Cat antibiotic resistance gene.

1. Digestion of pBAMI and Cat insert with the same restriction enzyme 2. Stop the digestion reaction by plasmid purification 3. Run a gel to check if pBAMI and the Cat insert have linearized 4. Calculate the volume of digested Cat insert and pBAMI needed for a 3:1 ratio for ligation 5. Use T4 DNA ligase to ligate pBAMI and the Cat insert 6. Run a gel to check if the ligated pBAMI with Cat has circularized 7. Perform a transformation and use the spread plate technique to plate pBAMI with Cat onto a LA/agar plate with chloramphenicol 8. Incubate plates at 37°C and then check for colony growth

Put the steps in correct order for cloning the pBlu plasmid with the Kan antibiotic resistance gene.

1. Digestion of pBLU and Kan insert with the same restriction enzyme 2. Stop the digestion reaction by plasmid purification 3. Run a gel to check if pBLU and the Kan insert have linearized 4. Calculate the volume of digested Kan insert and pBLU needed for a 3:1 ratio for ligation 5. Use T4 DNA ligase to ligate pBLU and the Kan insert 6. Run a gel to check if the ligated pBLU with Kan has circularized 7. Perform a transformation and use the spread plate technique to plate pBLU with Kan onto a LA/agar plate with kanamycin 8. Incubate plates at 37°C and then check for colony growth

Place the steps of the PCR reaction in order (1-6).

1. Initial DNA melting at 95°C 2. Cycling DNA melting at 95°C 3. Primers anneal at 55°C 4. Cycling extension by Taq polymerase at 72°C 5. Repeat the cycling steps 6. Final extension by Taq polymerase at 72°C

Put the steps in order (1-6) to ligate a DNA insert into a cloning vector.

1. Isolate plasmid from bacteria using the Qiagen kit 2. Digest plasmid and DNA insert with the same restriction 3. Deactivate/remove restriction enzyme to ensure digestion reaction has stopped 4. Confirm plasmid digestion by running digested plasmid on a gel 5. Combine digested plasmid and DNA insert with T4 DNA ligase for ligation 6. Confirm plasmid ligation by running product on a gel

You conducted a transformation with pBLU (contains the lacZ gene and has an ampicillin resistance gene in the backbone) that had a Cat insert ligated to disrupt the lacZ gene. Tell if there is growth and the color of the colonies on 1) a LA plate with ampicillin, 2) a LA plate with ampicillin and Xgal, 3) a LA plate with kanamycin, and 4) a LA plate with chloramphenicol.

1. LA plate with ampicillin: colony growth, white colonies 2. LA plate with Xgal and ampicillin: colony growth, white colonies 3. LA plate with kanamycin: no colony growth 4. LA plate with chloramphenicol: colony growth, white colonies

Place the steps for a transformation in order.

1. Thaw DH5α competent cells on ice 2. Add plasmid containing antibiotic resistance gene to competent cells and mix by tapping 3. Place competent cells with plasmid on ice for 30 minutes 4. Heat shock competent cells with plasmid at 42°C for 90 seconds 5. Place competent cells with plasmid back on ice for 2 minutes 6. Add SOC broth to competent cells with plasmid 7. Incubate competent cells with plasmid at 37°C for 1 hour with shaking 8. Plate competent cells with plasmid in SOC broth onto an agar plate with the correct antibiotic 9. Incubate at 37°C overnight and check for colonies

What is the molar ratio for a ligation reaction? Mae sure to indicate which part of the ratio is for the vector and which part is for the insert?

3 (insert):1 (vector)

You have digested 20ng/μL pBR322 plasmid with BamHI in a total volume of 10μL. You load the entire amount onto the column for the enzyme cleanup before a ligation reaction. You elute the clean plasmid with 40μL of elution buffer. What is the new concentration?

5ng/μL

Describe the difference between a single digest that will produce two bands on a gel and a double digest that produces two bands on a gel.

A single digest that has two bands on a gel uses only one restriction enzyme that cuts the plasmid twice to produces two bands. A double digest that produces two bands uses two restriction enzymes that cuts the plasmid once to produce two bands.

Which is not a part of the Codis STR loci for an individual's genetic fingerprint that the FBI established in 1996?

ALT

Choose an example of pseudoscience.

Alchemy

You have a plasmid with an unknown insert that could be for the ampicillin resistance gene (ampr), kanamycin resistance gene (kan), or chloramphenicol resistance gene (cat). You conduct a transformation of this plasmid with the unknown insert and plate it onto three separate LA plates: LA/Amp, LA/Kan, and LA/Chl. After an overnight incubation at 37°C, you see colonies appear only on the LA/Amp plate. What is the unknown insert (gene name needs written with correct formatting)?

Ampr

Describe how an uncut plasmid will look when run on a gel. Include the isoform names and where on the gel each isoform will appear.

An uncut plasmid will appear as three bands on the gel. The top band is the open circle isoform. The middle band is the linear isoform. The bottom band is the supercoiled isoform.

Which option is pseudoscience?

Astrology

A digestion of DNA with restriction enzymes can result in:

Blunt ends

Which is true regarding the steps of plasmid purification? All buffers need to be in the correct order with the correct mechanism of action.

Buffer N3 (denature protein)→ Buffer PB (stabilizes DNA onto silica membrane) → Buffer PE (washes DNA)

Match the buffer used in plasmid isolation with the correct mechanism (mode of action).

Buffer P2: 1. Alkaline Lysis Buffer PE: 2. Washes DNA Buffer P1: 3. Destabilizes outer membrane of bacterial cells Buffer EB: 4. Elutes DNA Buffer PB: 5. Tightens DNA bound to silica in the column Buffer N3: 6. Denature Proteins and precipitate formation

Which is true about chemical transformation?

Calcium ions are used to neutralize negatively charged molecules on the cell's outer membrane.

E. coli can take up DNA through:

Chemical Transformation, i.e. Calcium shock

Choose the correct answer on addressing antibiotic resistance genes and antibiotic use.

Chloramphenicol is an antibiotic

Describe the difference between the restriction digestion used during cloning and restriction digestion used for analysis of having an insert ligated into a plasmid. Use only one sentence describing cloning and one sentence for describing analysis.

Cloning: Restriction digestion is used to cut the plasmid and insert to give sticky ends to be used in the ligation reaction. Analysis: Restriction digestion is used to identify a unique cut pattern to show that the insert was added to the plasmid backbone.

Which is true about conjugation? Use the video and notes as references.

Conjugation involves many genes

Discuss what copy number is and give similarities and differences of low and high copy number plasmids.

Copy number is the number of plasmids in a cell. Both are used for replication inside a host and can be used for cloning or expression vectors. High copy number plasmids have low regulation stringency, while low copy number plasmids are more strictly regulated mechanisms for regulation. High copy number plasmids rely more on the host's machinery for replication, while low copy number plasmids have a DnaA-like enzyme encoded for on the plasmid.

In your own words (20 words or less), describe one advantage to using electroporation over chemical transformation.

Examples from lab manual: Works on bacteria that do not have a successful chemical transformation procedure; works in eukaryotic cells; transformation efficiency is not affected by size of DNA

List one way to identify articles as "bad science".

Examples such as cross reference the information, read the actual scientific article and not the news piece, understand characteristics of pseudoscience, etc.

List one danger of pseudoscience. Keep your answer to one sentence or less.

Examples such as misleading the public, having false data published, etc.

What step is involved in PCR?

Extension with Taq Polymerase

Write out the forward and reverse primer sequence that could be used for this template DNA strand. Make sure to include directionality. 5' - GGT TAA ATG GTC CCA TGG - 3'

Forward: 5' - GGT TAA ATG GTC CCA TGG - 3' Reverse: 5' - CCA TGG GAC CAT TTA ACC - 3'

Calculate the number of intermediate, total, and target strands formed during cycle 23 of PCR.

Intermediate: 64, Total: 8,388,608, Target: 8,388,544

You are ligating the kan insert (DNA insert) into a cloning vector. List the 3 possible outcomes/side reactions of how the kan gene will be inserted.

Kan gene inserted in the forward orientation, kan gene inserted in the reverse orientation, self-ligation of the plasmid (not inserted at all)

In your own words, list the three characteristics of a short tandem repeat.

Made of units of bases, two to five bases long, multiple repeats

A transformation was conducted, and the research forgot to conduct a heat shock step. How would you expect the LA plate to look after an overnight incubation? Keep your answer to less than 10 words.

No growth/colonies

Which is true about cutting a circular plasmid once with one restriction enzyme?

One linear fragment is created

Choose an option to make this title more accurate and less sensationalized. See the article for reference: https://www.bbc.com/news/health-53137328 (You do not need access to the article to answer the question). "Cancer drug: New treatment halts tumour growth"

Phase I trial of Berzosertib results in slowing tumor growth when given with chemotherapy"

Which is not a rule for designing primers?

Primers need to have secondary structure in order to anneal correctly to template DNA

Name the type of enzyme used in a restriction digestion reaction and PCR respectively. Make sure to identify which enzymes belongs to which reaction.

Restriction digestion: Restriction enzymes; PCR: DNA Polymerase (Taq Polymerase)

Which group of enzymes are used restriction digestion reactions?

Restriction enzymes

What is the purpose of using restriction enzymes on a purified plasmid?

Restriction enzymes are used for digesting a plasmid for cloning.

Which is true about restriction enzymes?

Restriction enzymes cut at a recognition sequence.

What is the difference between restriction and ligation reactions? Keep your answer to 3 sentences

Restriction is the cutting of double-stranded DNA. Ligation is the joining of pieces of DNA.

What enzyme is used for ligation reactions, and what is the molar ratio of insert to plasmid that is needed?

T4 DNA Ligase, 3:1

In most instances of cloning, a high copy number plasmid is advantageous to use. Name one reason that you would want to use a low copy number plasmid instead of a high copy number plasmid. Use your lab manual and PowerPoint as a reference Keep your answer to 3 sentences.

The DNA insert cloned into the plasmid codes for a protein that is toxic in high amounts to the bacteria.

You conduct a ligation reaction after a restriction enzyme cleanup and run a gel to check that the ligation occurred properly. You observe a band at the size of the linearized plasmid and the size of the insert. What caused this result?

The T4 DNA ligase was not added to the reaction.

You are running a plasmid purification, and you accidentally use buffer PB instead of buffer EB. What will happen to the plasmid? Keep your answer to less than 10 words.

The plasmid will not be eluted off the column.

What would happen if you had too many G's and C's in your primer?

The primer could potentially self-bind if to many G's and C's at the 5' and 3' end.

Which is true about polymerase chain reaction (PCR)?

The temperature for melting of DNA for denaturation occurs around 95°C

Which is not true about short tandem repeats (STRs)?

They come from your mother only.

You have designed the primer pair below for an experiment. Tell if this is an acceptable primer set to use. Discuss melting temperature (Tm), G/C content, and length. Provide no more than one sentence for each topic of discussion (6 sentences maximum). Forward: 5'- GCA AAT TAT GCA ACC GCC -3' Reverse: 5'- GAT TTA CGC TTA TTT TGC TAA CAA GAA TTG G -3'

This primer set is not acceptable to use. The Tm for the forward is 54°C (in range), and the Tm for the reverse is 82°C (out of acceptable range). The primers are not within 5°C of each other. The G/C content is in range for the forward primer at 50% and is out of range for the reverse primer at 32%. There are too many (3bp) G/C's at the 3' end of the forward primer. The length of the forward primer is in range at 18bp, but the length of the reverse primer is not in range at 31 bp.

Label the statements on restriction enzymes- True or False.

True: Restriction sites can be palindromes True: The restriction site is where the cut occurs False: Restriction enzymes are only found in bacteria True: Restriction enzymes can provide bacteria defense against foreign DNA False: Restriction enzymes do not naturally occur in bacteria or archaea

Which is not a step in the cloning process?

Use T4 DNA ligase to digest the DNA insert and cloning vector

Calculate the volume of plasmid and insert needed for a ligation reaction using the specifications for NEBL T4 DNA ligase (0.020 pmol vector, 0.060 pmol insert). This reaction is ligating a 3kb vector (25ng/μL) and a 2kb (10ng/μL) insert together, and the fragments have complementary sticky ends. Round final answers to the nearest hundredths place.

Vector: 1.59μL, Insert: 7.93μL

What is true about blue/white screening technology?

When the LacZ gene is intact, and the media has Xgal, colonies that form will appear blue

Choose the correct expected result regarding antibiotic resistance when plating plasmids with inserts.

When the pBR322 plasmid with the ampicillin resistance gene (Ampr) is plated onto an agar plate with ampicillin, there are colonies present

You have run a gel to check to see if your ligation reaction has been completed before moving on to conducting a transformation. You observe that you have a band the size of the plasmid on your gel (plasmid is still linear). What has happened?

You did not conduct a cleanup step and the restriction enzyme used is still able to cut your plasmid.

Explain how you would obtain two bands on a gel using a single digest.

You would obtain two bands on a gel from a single digest if there were two recognition sites for the restriction enzyme on the circular plasmid.