MCB4034L All Quizzes

What statement describes a plasmid?

Extra-chromosomal circular DNA

Which of the options below represents the typical size of a plasmid. Pay attention to the units!

4 kb

Today we will isolate plasmid DNA from a bacterial culture. We will be using the Zyppy plasmid isolation kit from Zymo. The handouts and the manufacturer's protocol are provided as part of the material posted under Modules > Week 1. You will need both to answer this question.

The basic protocol provided by Zymo (pdf) is based on processing 600 microliter (0.6 mL) of the bacterial culture. Instead, we will harvest the cells by three consecutive centrifugation steps using 1.5 mL culture each time. Select from the answers below the one correct reason why we use this larger volume.

We need to isolate more plasmid DNA for the subsequent experiments than the volume of culture listed in the standard protocol would yield

It is important to operate centrifuges with balanced rotors to avoid strain on the motor and internal parts. The most common configuration for small centrifuges is a rotor that can accommodate 12 tubes.

The choices below represent the number of tubes you need to spin in the centrifuge. Each tube has the same mass. Select which of the answer(s) represents a situation where you absolutely HAVE to use a dummy tube (a centrifuge tube filled with water to match the mass of the tube with your sample). There may be more than one correct answer.

1

During the first day of class, Dr V talked about recombinant proteins. Which of the following statements correctly describes a recombinant protein? There may be more than one correct answer.

- A recombinant protein can be expressed in an organism that normally (in nature/wild-type state) does not have the ability to produce that protein

- A recombinant protein is encoded by an engineered ("designed") DNA sequence

Mia is going to isolate DNA from a 1.2% agarose gel using the QiaQuick Gel Extraction kit. In a 1.5 mL tube she collects a gel slice with a mass of 120 mg. How much QG buffer will she need to add to this gel slice according to the protocol available under Modules > Week 2? Pay attention to the units.

360 microliter

This question relies on the same protocol as the previous question. In order to maximize the DNA concentration of the gel-extracted DNA, what is the volume you need to use for the elution step?

30 microliter

What is the natural function of a restriction enzyme?

It cuts bacteriophage DNA that has entered the bacterial cell

You have learned that restriction enzymes recognize palindromic sequences. Which of the following DNA sequences is a likely restriction site, when it is present in double-stranded DNA? Multiple answers may be correct!

- 5'-AAGCTT-3'

- 5'-CCCGGG-3'

Restriction enzymes will generate either blunt or sticky ends. Which of the following statements are correct? There may be more than one correct answer.

- Sticky ends are also referred to as "overhanging ends"

- Any two blunt-ended DNA fragments can be joined, even if those DNA fragments were generated by different restriction enzymes

- "Sticky ends" are called sticky because they can stick together via hydrogen bonds

During today's lab we will set up a ligation reaction. What do the enzyme DNA ligase do?

It forms phosphodiester bonds to join two DNA fragments

For the ligation reaction we are combining the plasmid and insert in a 1:2 molar ration. What is the rationale behind this particular ratio?

- A greater proportion of insert molecules in the reaction would result in the formation of concatemers

- A greater proportion of plasmid in the reaction would reduce the # of plasmid molecules that will incorporate an insert

The concentrations of the plasmid and insert we use in the ligation are based on the absorbance of UV light measured with a spectrophotometer. Which one of the following statements about the absorbance values is correct?

When the A260/A280 ration is between 1.8 and 2.0, we consider the DNA clean

After the ligation we will transform bacteria. What is needed to transform E. coli bacteria:

- E. coli competent cells

- A plasmid harboring an origin of replication

You need to design primers for your next experiment and are constrained by the position of the primers in GC-rich regions of the template DNA. After considerable effort, you have come up with two possible primer pairs, listed below, neither or which is ideal.

Primer pair 1: Upper primer U1 can form a 3' heterodimer with lower primer L1; this dimer has a Tm of 67C

Primer pair 2: Upper primer U2 can form a 5' homodimer; this dimer has a Tm of 64C

With primer pair 2 you will have a greater likelihood of producing the desired product than with primer pair 1

Serina is going to use PCR to amplify a 1-kb fragment of DNA. She is editing a 3-step program in the thermal cycler. What is the appropriate parameters for the recurring extension step?

1 minute @ 72C

Today we will select colonies that grew from individual E. coli cells in the tubes to which we added 4 uL ligation reaction. After we let the cells sit on ice for 10 min., we let the cells recover in SOC medium at 37C, and spread the cells on solid medium (beads!) for overnight growth. Given what you have learned about the plasmid we are using in MCB4034L, how did we make sure that only E. coli cells that have taken up a plasmid molecule survived?

We used medium containing kanamycin

PCR relies on a thermostable DNA polymerase replicating template DNA delineated by two primers. What is the template for our PCR experiment today?

The recombinant plasmid

Select the correct statement(s) about colony PCR from the following options. There may be more than one answer.

- I can distinguish a plasmid with a single insert from a plasmid w/o insert based on the size of the PCR product

- I can distinguish a plasmid w/ a single insert from a plasmid w/ concatemers based on the size of the PCR prodcut

Taq DNA polymerase makes mistakes (insertion, deletion, wrong base incorporated) at an average rate of 1 per 1,000 nucleotides synthesized. Such mistakes can have negative impacts when the goal is to create a recombinant protein.

True or False: "For our experiment we don't need to be concerned about mistakes made by Taq DNA polymerase, because the CBM cDNA sequence we amplified is only 250 basepairs in length."

False

Review question: MacKenzie needs to amplify a 1.5 kb target sequence during her colony PCR. She is going to use a 3-step program. What is the correct combination of temperature and time for the extension step of the PCR?

90 sec at 72C

At neutral pH, DNA has a __________, and when it is subjected to agarose gel electrophoresis, the _________ fragments will migrate through the gel the fastest.

negative; smallest

What are the correct answers relating to colony PCR?

- Colony PCR enables me to identify plasmids containing concatemers, which are undesirable

- A benefit of colony PCR is that, on average, I end up isolating plasmids from a smaller number of cultures compared to the alternative procedure that does not include colony PCR

- Colony PCR enables me to identify plasmids w/o inserts, which are undesirable

This question and the next two relate to the Zyppy plasmid isolation procedure, which we will use again as we did during Week 2 of the course.

We will start with the 5 mL cultures you inoculated on Thursday last week. Just like in week 2, we will use 3 x 1.5 mL culture rather than the 0.6 mL listed in the manufacturer's standard protocol. Why?

Using only 0.6 mL of culture will not yield enough plasmid to enable sequencing

After adding the neutralization buffer to the lysed cells, a curdled mass of cell debris is formed. This lump is separated from the liquid via centrifugation. What do you do with the liquid fraction (supernatant)?

Carefully remove it from the centrifuge tube with your pipette and load it on the spin column

During the last step of the protocol, elution buffer is added to the spin column, and the spin column, while sitting in a 1.5 mL tube, is centrifuged. Where is the plasmid when you are finished centrifuging?

In the bottom of the 1.5mL collection tube

Review question: Maya needs to set up a ligation involving a 4 kb plasmid and a 500 bp PCR product. The molar ratio needs to be 1 plasmid : 2 insert. The protocol specifies the use of 100 ng plasmid DNA. How much insert DNA does Maya need to add?

25 ng

Review question: You need to prepare an enzymatic reaction involving a 4x concentrated buffer. The final (=total) volume of the reaction is 20 uL. What is the correct volume of 4x buffer you will need to pipette in the reaction tube?

5 uL

One of the sequenced recombinant plasmids was introduced in the expression strain E. coli named BL21(DE3)pLysS, to enable production of the recombinant GFP-CBM protein. The cells don't make the protein unless the culture is induced. What was done to induce the cells?

IPTG was added to an established culture

Nirvana needs to determine the mass of a cell pellet she obtained after centrifuging 35 mL bacterial culture in a 50 mL tube. The pellet needs to stay in the tube. How can she quickly determine the bacterial mass?

She needs to weigh the tube with the pellet + subtract the mass of the empty tube that was recorded beforehand

After Nirvana has established that her 35-mL culture yielded a cell pellet of 0.3 g, what is the recommended volume of BugBuster Lysis reagent for this amount of bacterial cells?

1.5mL

The E. coli expression strain BL21 (DE3) is available in several variants. One of those contains the pLysE plasmid. Why would you opt for this particular version when you want to express a recombinant protein that does not exist in nature? Select the correct answer from the choices below.

In combination with a plasmid containing the lac operator, this strain will be the best choice when you have serious concerns about the cytotoxicty of the recombinant protein to the E. coli host cells.

Cam has cloned a cDNA fragment in a plasmid and wants to produce the recombinant protein encoded by this cDNA fragment. The cDNA contains an open reading frame (ORF) of 1,140 bp.

Select from the choices below the correct answer relating to the molecular weight of the recombinant protein this cDNA fragment encodes.

The recombinant protein has a predicted molecular weight of 41.8 kDa

Today we will be performing SDS-polyacrylamide gel electrophoresis of proteins. What is the purpose of the SDS?

It is a detergent that associates with the proteins and ensures all proteins have a negative charge, regardless of their isoelectric point.

While there are some similarities between SDS polyacrylamide gel electrophoresis of proteins and agarose gel electrophoresis of DNA, there are also some clear differences. Which of the following options represent actual differences? Multiple answers may be correct.

- Protein samples are denatured prior to and during SDS-PAGE, whereas DNA in agarose gels is not

- During electrophoresis agarose gels are placed in a horizontal position, polyacrylamide gels in a vertical position

- Polyacrylamide gels are typically subjected to a considerably higher voltage than agarose gells

Maha is preparing two protein samples for PAGE. She is supposed to load 15 uL of a crude lysate on the gel, and 6 uL of purified protein. She has aliquoted these volumes in two labeled tubes and has to add the sample loading dye (a.k.a. loading buffer) to these two samples. The dye comes as a 4x concentrate. How much loading dye should she add to the 15 uL of crude lysate and how much to the 6 uL of purified protein?

She should add 5uL sample loading dye to sample A, and 2 uL to sample B to ensure the loading dye to diluted correctly

What is Maha supposed to do after she has added the correct amount of loading dye (a.k.a. loading buffer) to her samples?

Heat the samples in a heating block at 95C for 5 min., then immediately transfer them to ice, and then load them on the gel

Which one of the methods listed below is commonly used to visualize proteins in polyacrylamide gels?

Coomassie Brilliant Blue

Below is the image of a polyacrylamide gel with a number of different protein samples that have been stained with a dye. The lanes are numbered. Select from the answers below the lane(s) with a protein sample that has been purified to apparent homogeneity. There may be more than one correct answer.

Lane 8 (one clear blue mark)