MOLBIO LAB 7

In any amplification experiment, which procedure is considered the first, pre-analytical step?

Nucleic acid extraction

What is the primary objective of the cell lysis step in nucleic acid extraction?

To release nucleic acids from the cell and denature nucleases

Which reagent is typically used to precipitate nucleic acids out of a solution?

Ethanol

In solid-phase extraction, nucleic acids bind to a membrane surface typically made of _____.

Silica

In Solid Phase Isolation, the DNA is isolated or located in what area?

Silica Membrane

What is the specific purpose of the washing steps during DNA purification?

To remove unbound substances such as proteins, cell debris, and PCR inhibitors

Which process involves removing purified nucleic acid from a membrane using water or a low-salt buffer?

Elution

What is the product after the initial cell lysis step in DNA extraction?

Lysate

What is the term for the final product containing DNA after the elution step?

Eluate

Which two organic reagents are used in the organic isolation method to form two distinct layers?

Phenol and Chloroform

In an organic DNA isolation, which specific layer contains the DNA?

The upper aqueous (hydrophilic) layer

In organic DNA isolation, what substances are found in the bottom hydrophobic layer?

Lipids and proteins

Why must a rehydration buffer like Tris-EDTA be added to DNA after ethanol precipitation?

DNA is insoluble in ethanol and must be dissolved for use.

The inorganic isolation method is also commonly known as the _____ method.

Salting-out

How does the salting-out method selectively precipitate proteins?

By using low pH and high salt conditions

Which alcohol is used in the inorganic isolation method to precipitate DNA?

Isopropanol

According to Meijer et al., what is a primary disadvantage of the phenol-chloroform method?

It causes significant DNA loss

What is a common biological source for the silica particles used in solid-phase extraction?

Diatomaceous earth

Name one chaotropic salt used to promote the absorption of nucleic acids to silica.

Guanidine hydrochloride (or Guanidine isothiocyanate, Sodium iodide, or Sodium Perchlorate)

In the solid-phase procedure, what is the 'eluate'?

The pure DNA sample collected after the elution step

What is the surface charge of magnetic beads used to bind proteins and cellular debris selectively?

Negative surface charge

Give an example of an automated extraction instrument that utilizes magnetic beads.

The miniMag (bioMerieux) or MagNA Pure (Roche)

What is a major limitation of manual extraction methods regarding PCR testing?

Residual ethanol can inhibit the PCR process

What must be added to PW Buffer and Wash Buffer before they can be used in extraction?

Absolute ethanol

What is the maximum number of bacterial cells suggested for harvesting?

2×10^9 cells.

What is the function of Proteinase K?

It targets and digests proteins in the cell membrane

What solution targets the protein in the cell membrane?

Proteinase K solution

What is the required incubation temperature for the Gram-negative bacteria lysis step?

56 degrees Celsius

During the 1-hour incubation for lysis, how often should the sample be vortexed?

Every 30 minutes

What enzyme is added to the lysate to ensure the sample is specifically 'DNA free of RNA'?

RNase A

What is the centrifugal force and duration required for the 'Drying Step' in the spin column procedure?

12,000×g for 2 minutes

What is the recommended storage temperature for isolated DNA if it is not tested on the same day?

-20 degrees Celsius

Which gene is targeted to identify E.coli?

16S rRNA

Which reagent is used as the 'digesting solution' to resuspend the bacterial pellet?

TES buffer

What are the two chemical components that make up the TES buffer?

Tris-EDTA & Sodium Dodecyl Sulfate (SDS)

During the DNA purification washing steps, what is done with the 'flow-through' after centrifugation?

It is discarded

What volume of elution buffer is typically used in the final step of the isolation protocol?

100 uL

What is the nucleic acid extracted?

Genomic DNA