Lecture 4

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Questions and Answers

What is the primary purpose of cell lysis in a gDNA extraction protocol?

  • To isolate and pellet proteins
  • To break open cells and release DNA (correct)
  • To prepare DNA for precipitation
  • To precipitate proteins for easy removal

Which of the following cell lysis methods is specifically suited for halophilic organisms?

  • French Press
  • Boiling
  • Osmotic shock (correct)
  • Bead beating

What is the role of EDTA in chemical cell lysis?

  • It disrupts cell membranes.
  • It degrades peptidoglycan in Gram-positive bacteria.
  • It chelates $Mg^{2+}$, leading to LPS release and outer membrane detachment. (correct)
  • It denatures proteins.

Why is protein precipitation a necessary step in gDNA extraction?

<p>To isolate proteins, removing them as contaminants from the DNA sample (B)</p> Signup and view all the answers

In protein precipitation, what is the principle behind 'salting out' proteins?

<p>Salts disrupt the hydrogen bonds between proteins, causing them to aggregate and precipitate. (C)</p> Signup and view all the answers

What is the purpose of adding ethanol (EtOH) during DNA precipitation?

<p>To deplete the water hydration shell around DNA, causing it to precipitate (C)</p> Signup and view all the answers

How do cations (e.g., $NH_4^+$) contribute to DNA precipitation?

<p>By forming stable bonds with the DNA's phosphate groups ($PO_4^{3-}$), neutralizing the charge (B)</p> Signup and view all the answers

What is the function of Alkaline Phosphatase in gDNA extraction?

<p>It removes 5'-phosphate groups from the DNA terminus. (A)</p> Signup and view all the answers

Which enzyme is used to digest agarose when isolating a specific DNA amplicon from a gel?

<p>β-Agarase I (C)</p> Signup and view all the answers

Why is Alkaline Protease used during gDNA extraction?

<p>To inactivate endonucleases released during cell lysis (C)</p> Signup and view all the answers

What is the role of ammonium sulfate ($NH_4SO_4$) in gDNA extraction?

<p>It precipitates proteins. (B)</p> Signup and view all the answers

Isopropanol is used in gDNA extraction for what purpose?

<p>To precipitate DNA (C)</p> Signup and view all the answers

What is the recommended solution for long-term storage of extracted DNA?

<p>A buffered solution (e.g., 10 mM Tris-Cl, pH 8.5) (B)</p> Signup and view all the answers

In an old-school DNA extraction method using Phenol:Chloroform:Isoamyl Alcohol, what is a major disadvantage?

<p>It requires special waste removal due to the use of nasty compounds. (A)</p> Signup and view all the answers

In the Nanodrop readings, what does the 260/280 ratio indicate, and what is the expected value for pure DNA?

<p>DNA and RNA purity; ~1.8 (D)</p> Signup and view all the answers

A Nanodrop reading for a DNA sample shows a 260/230 ratio significantly lower than ~2-2.2. What does this indicate?

<p>Possible contamination (A)</p> Signup and view all the answers

In the context of the V. fischeri genomic DNA extraction, what is the ultimate goal?

<p>To obtain many copies of <em>lux</em> genes (A)</p> Signup and view all the answers

In the project snapshot, the purified V. fischeri genomic DNA containing lux genes is digested with a restriction enzyme. What is the next step in creating a bioluminescent E. coli?

<p>Joining the lux genes with a plasmid using DNA ligase (C)</p> Signup and view all the answers

After creating a recombinant plasmid containing lux genes, what is the next step to produce bioluminescent E. coli?

<p>Transform <em>E. coli</em> with the plasmid (C)</p> Signup and view all the answers

After transforming E. coli with a plasmid containing lux genes, what is the method of identifying/isolating successful transformants?

<p>Observing <em>E. coli</em> growth on a medium containing ampicillin (A), Observing <em>E. coli</em> for bioluminescence (B)</p> Signup and view all the answers

Which of the following is NOT a main step involved in chromosomal gDNA extraction?

<p>RNA Amplification (B)</p> Signup and view all the answers

Which mechanical method of cell lysis involves using high pressure to disrupt cell membranes?

<p>French Press (A)</p> Signup and view all the answers

Which of the following chemical substances disrupts cell membranes and denatures proteins during cell lysis?

<p>SDS (A)</p> Signup and view all the answers

Lysozyme is an enzymatic agent used in cell lysis that targets which specific component of bacterial cells?

<p>Peptidoglycan (A)</p> Signup and view all the answers

According to the information provided, which of the following is NOT a characteristic or component associated with the old-school DNA extraction method using Phenol:Chloroform:Isoamyl Alcohol (25:24:1)?

<p>Safe and environmentally friendly (A)</p> Signup and view all the answers

Regarding the process of protein precipitation during gDNA extraction, what role do hydrogen bonds (H-bonds) play in maintaining proteins in a soluble, aqueous solution?

<p>H-bonds allow interactions between the protein's R-groups, protein backbone, and water (H2O) (A)</p> Signup and view all the answers

Which of the following best details the effects of adding EtOH to a solution during the process of DNA Precipitation?

<p>EtOH depletes the H2O hydration shell surrounding DNA (C)</p> Signup and view all the answers

How do free cations contribute to the neutralization of charges on DNA molecules therefore aiding in DNA precipitation?

<p>Free cations form stable bonds with the DNA's $PO_4^{3-}$ (D)</p> Signup and view all the answers

What is the specific function of DNase I in the chromosomal gDNA extraction process?

<p>Hydrolyzes phophodiester bonds (A)</p> Signup and view all the answers

According to the information provided, in which scenario would performing digests using β-Agarase I be useful?

<p>To digest agarose and isolate one amplicon out from a gel (B)</p> Signup and view all the answers

What role does alkaline protease play in the chromosomal gDNA extraction process?

<p>It inactivates endonucleases (C)</p> Signup and view all the answers

Which statement effectively outlines the impact of increased ionic strength on the interactions between protein and water (H2O)?

<p>Increased ionic strength decreases protein solubility in H2O (D)</p> Signup and view all the answers

Why is a buffered solution required for the long-term storage of extracted DNA?

<p>To maintain the pH to protect the nucleic acids over time (B)</p> Signup and view all the answers

What is the catalog number and lot number important to record when following the Genomic Prep Protocol?

<p>To track and verify the materials that were used (C)</p> Signup and view all the answers

During a project in which V. fischeri is used to transform E. coli, what is being transformed into it?

<p>Plasmid (B)</p> Signup and view all the answers

What is the purpose of inserting the lux genes into E. coli in the project snapshot?

<p>To enable <em>E. coli</em> to become bioluminescent (A)</p> Signup and view all the answers

In the depiction of E. coli and V. fischeri, what is suggested by the presence of 'Ampicillin' in the E. coli test tube?

<p>That E. coli is resistant to ampicillin (B)</p> Signup and view all the answers

Which of the following is the final step in the project snapshot?

<p>Screen for E. coli colonies with plasmid (B)</p> Signup and view all the answers

Flashcards

Cell Lysis

The process of breaking open cells to release their contents, including DNA.

Cell Lysis Methods

Mechanical, chemical, and enzymatic methods used to break open cells for DNA extraction.

Bead Beating

A mechanical method of cell lysis using tiny beads to physically disrupt cell walls.

French Press

A mechanical method of cell lysis using high pressure to force cells through a small space.

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Freeze/Thaw

Mechanical lysis via repeated freezing and thawing to disrupt cell structure.

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Boiling

Cell lysis by heating cells until they burst open.

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Osmotic Shock

Cell lysis due to osmotic pressure differences, used only for halophilic organisms.

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SDS

Chemical lysis using SDS to disrupt the cell membrane and denature proteins.

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EDTA

Chemical lysis using EDTA to chelate Mg2+, leading to LPS release and membrane detachment.

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Lysozyme

Enzymatic lysis via lysozyme, which degrades peptidoglycan in Gram-positive bacteria.

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Protein precipitation

Isolating proteins away from everything else which allows for easy removal of these undesirables.

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DNA precipitation

Removing other 'stuff' in the solution besides DNA by pelleted and poured off.

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Phenol-Chloroform Extraction

A DNA extraction method using phenol, chloroform, and isoamyl alcohol.

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Protein Precipitation

Using salt to 'salt out' or precipitate proteins from a solution.

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Ethanol Precipitation

Adding ethanol to reduce water polarity and precipitate DNA.

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Alkaline Phosphatase

An enzyme that removes 5'-PO₄³⁻ groups from the DNA terminus.

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DNase I

An enzyme that breaks phosphodiester bonds in DNA.

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Beta-Agarase I

An enzyme that breaks down agarose.

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Alkaline Protease

An enzyme to inactivate endonucleases and other contaminating proteins.

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Ammonium Sulfate

A salt used to precipitate proteins by altering water solubility.

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Isopropanol

An alcohol used to precipitate DNA out of solution.

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Storing DNA

For the short-term, sterile water is okay; for long-term, a buffered solution is required.

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Nanodrop

A device that measures the concentration and purity of nucleic acids in a sample.

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260/280 Ratio

Indicates DNA and RNA purity. Pure DNA is ~1.8, pure RNA is ~2.0

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260/230

Indicates Nucleic acid purity. Pure DNA is ~2-2.2. Lower values may indicate contamination

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Study Notes

  • Critical Analysis 1 is due February 7th
  • Follow the rubric when completing Critical Analysis 1

Chromosomal gDNA Extractions

  • Three main steps involved in chromosomal gDNA extraction are cell lysis, protein precipitation, and DNA precipitation

Cell Lysis Methods

  • Cell lysis involves breaking open the cells to release the DNA
  • Mechanical methods include bead beating, French press, freeze/thaw, and boiling
  • Chemical methods include osmotic shock (for halophilic organisms), SDS (disrupts cell membranes and denatures proteins), and EDTA (chelates Mg2+, leading to LPS release and outer membrane detachment)
  • Enzymatic method involves using lysozyme, which degrades peptidoglycan in Gram-positive bacteria

Old School DNA Extraction Method

  • Phenol: Chloroform: Isoamyl Alcohol (25:24:1) is still effective
  • The method uses nasty compounds and requires special waste removal

Protein Precipitation

  • Precipitation is salting out proteins
  • Proteins are maintained in solution when hydrogen atoms can form H-bonds between the R-groups, protein backbone, and H₂O

DNA Precipitation

  • EtOH polarity is less than H₂O polarity
  • Adding EtOH depletes the H₂O hydration shell surrounding DNA
  • PO43- groups in the DNA then become more exposed
  • Free cations (e.g., NH4+) form stable bonds with the DNA's PO43- and neutralize the charges
  • DNA is then precipitated

Chemicals and Their Functions

  • Alkaline Phosphatase removes 5'-PO43- from the DNA terminus
  • DNAse I hydrolyzes phosphodiester bonds in dsDNA and ssDNA
  • ß-Agarase I digests agarose and is useful in isolating one amplicon out from a gel
  • Alkaline Protease inactivates endonucleases and other proteins released during cell lysis
  • Ammonium Sulfate precipitates proteins out of solution due to their high solubility in H₂O and increased ionic strength
  • Isopropanol helps precipitate the DNA out of solution

Storing DNA

  • For short-term storage, sterile water is suitable
  • For long-term storage, a buffered solution is required for DNA stability, such as 10 mM Tris-Cl, pH 8.5

Lab Protocol

  • The objective is to obtain gDNA of A. fischeri, and large amounts of lux copies are desired
  • A Genomic Prep Protocol should be followed using a Promega Wizard gDNA Purification Kit
  • The catalog and lot numbers should be recorded

Nanodrop

  • Follow the protocol and wipe it down after
  • Three numbers that matter in Nanodrop results are concentration, 260/280, and 260/230
  • Concentration is measured in Ng/ul
  • The 260/280 ratio indicates DNA and RNA purity, with pure DNA at ~1.8 and pure RNA at ~2.0
  • The 260/230 ratio indicates nucleic acid purity, with pure DNA at ~2-2.2; lower values may indicate contamination

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