Sample Preparation & Nucleic Acid Extraction Flashcards

What is the primary role of guanidinium thiocyanate in many nucleic acid lysis buffers?

Answer: It denatures proteins, inactivates nucleases, and promotes nucleic acid binding to silica

Guanidinium salts are chaotropic agents that denature proteins including RNases and enable nucleic acids to adsorb to silica.

Under which conditions does DNA bind most efficiently to a silica spin column membrane?

Answer: High concentration of chaotropic salt

High chaotropic salt dehydrates nucleic acids and the silica surface, allowing binding, while low-salt buffers are used for elution.

Why are ethanol-based wash buffers used during silica column extraction?

Answer: To remove salts and contaminants while keeping nucleic acids bound

Ethanol keeps nucleic acids insoluble on the membrane while washing away salts, proteins, and chaotropes.

A lab notices PCR inhibition after column extractions; the technician skipped the final empty 'dry' spin. What is the most likely cause?

Answer: Carryover of residual ethanol into the eluate

The dry spin removes residual ethanol, which can inhibit polymerase activity if carried into the eluate.

What is the function of Proteinase K during sample lysis?

Answer: It digests proteins, including nucleases and histones, to release nucleic acids

Proteinase K is a broad-spectrum serine protease that degrades proteins and nucleases, helping release intact nucleic acids.

In acid phenol-chloroform extraction (pH ~4.5), where does RNA partition?

Answer: Aqueous phase

At acidic pH, DNA partitions to the interphase/organic phase while RNA remains in the aqueous phase.

Compared with ethanol, what is a key characteristic of isopropanol for nucleic acid precipitation?

Answer: It requires a smaller volume (0.7-1 volume) but tends to co-precipitate more salt

Isopropanol precipitates DNA at lower volumes, but salts are less soluble in it, so a 70% ethanol wash is typically needed afterward.