reagent study biol 101

What reagent is used in Lab 1 PCR to provide the correct salt concentration and pH? What concentration is used? What happens if omitted?

1X PCR buffer. Maintains proper salt concentration and pH for Taq polymerase. Without it, the polymerase loses activity and PCR fails.

What reagent is used in Lab 1 as the essential cofactor for Taq polymerase? What concentration is used? What happens if omitted?

MgCl2 (1–2.5 mM). Required cofactor for DNA polymerase. Without Mg2+, DNA synthesis does not occur.

What reagent provides the building blocks for DNA synthesis in Lab 1? What happens if omitted?

dNTPs. They are the DNA monomers. Without them, no DNA can be synthesized.

What reagent is used to bring PCR reagents to their final concentrations? What happens if omitted?

ddH2O. Sterile water used to adjust final concentrations. Without it, reagent concentrations become too high and the reaction is disrupted.

What reagent in Lab 2 contains RNase A? What concentration/components? What happens if omitted?

Buffer P1 (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 µg/mL RNase A). RNase removes RNA. Without it, RNA contaminates plasmid DNA.

What reagent is the lysis buffer in Lab 2? Components? What happens if omitted?

Buffer P2 (1% SDS, 0.2 M NaOH). Lyses cells and denatures DNA/proteins. Without it, cells won't lyse.

What reagent neutralizes the lysate in Lab 2? Components?

Buffer N3 (3.0 M potassium acetate, pH 5.5). Neutralizes NaOH so plasmid DNA renatures while genomic DNA/proteins precipitate.

What reagent is the wash buffer in Lab 2 that removes salts? What happens if omitted?

Buffer PE. Removes salts. Without it, salts interfere with downstream reactions.

What reagent elutes plasmid DNA in Lab 2? Concentration?

Buffer EB (10 mM Tris-HCl, pH 8.5). Elutes DNA from silica. Without it, DNA stays bound.

What reagent provides optimal conditions for restriction enzymes in Lab 3?

10X rCutSmart buffer. Provides optimal salt and pH. Wrong buffer prevents digestion.

What reagent makes DNA visible in Lab 3? Concentration?

Ethidium bromide (25 µL of 10 mg/mL stock per 500 mL gel). Intercalates DNA and fluoresces under UV.

What reagent dissolves agarose during gel extraction? Concentration/pH?

Buffer QG (guanidine thiocyanate, pH ~6.6). Dissolves gel and promotes DNA binding to silica.

What reagent provides energy for ligation in Lab 4?

10X Ligase Buffer containing ATP. ATP is required by DNA ligase to seal phosphodiester bonds.

What reagent induces protein expression in Lab 5? Concentration?

IPTG (1 mM). Induces T7 RNA polymerase expression by releasing the lac repressor.

What reagent binds His-tagged proteins in Lab 5?

MagneHis Ni particles. Nickel binds the 6xHis tag for purification.

What reagent elutes His-tagged proteins in Lab 5? Concentration?

Elution buffer containing 500 mM imidazole. Competes with His tag for nickel binding.

What reagent allows RNA to bind the silica column in Lab 6?

95% ethanol. Creates conditions for RNA binding. Without it, RNA flows through.

What reagent is used to synthesize cDNA in Lab 6? Concentration?

Oligo-dT primer (50 µM, 23-mer). Provides a primer for reverse transcriptase.

What reagent denatures proteins and gives them a uniform negative charge in Lab 7?

SDS (0.1% in running buffer). Denatures proteins and equalizes charge-to-mass ratio.

What reagent reduces disulfide bonds in Lab 7?

DTT or β-mercaptoethanol. Breaks disulfide bonds for complete denaturation.

What reagent blocks nonspecific antibody binding in Lab 8?

Blocking buffer (5% milk in TBS-T). Prevents antibodies from binding nonspecifically.

What reagent is the primary antibody in Lab 8?

Mouse anti-His antibody. Binds specifically to the 6xHis tag.

What reagent generates light in Lab 8?

Luminol (ECL). HRP converts it into light for chemiluminescent detection.

What reagent unfolds proteins in Lab 9? Concentration?

Urea (6 M stock, 3 M final). Denatures proteins before trypsin digestion.

What reagent digests proteins into peptides in Lab 9?

Trypsin (1 mg/mL). Cleaves after lysine and arginine residues.