Q.43. A PCR in a 𝟏𝟎𝟎𝝁 𝐋 reaction volume, containing two primers at a concentration of 𝟎. 𝟐𝝁𝐌 each,
is set up to amplify a 250 base pair DNA fragment. Consider the average molecular weight of one base
pair as 660 Da . If the primers are fully consumed by the end of the reaction, the amount of the final PCR
product formed is ____ 𝝁𝐠 (rounded off to one decimal place).

PCR reactions amplify specific DNA fragments, with product yield often limited by primer availability. When primers are fully consumed, the final product amount for a 250 bp fragment in a 100 _μL reaction with 0.2 _μM each primer calculates to 6.6 _μg.

Step-by-Step Calculation

Each primer at 0.2 _μM in 100 _μL (0.1 mL) equals 0.02 nanomoles, so two primers total 0.04 nanomoles or 4×10^-11 moles of product formed.

The 250 bp product’s molecular weight is 250×660=165,000 g/mol.

Mass equals moles times MW: 4×10^-11×165,000=6.6×10^-6 g or 6.6 _μg.

Why Primers Limit Yield

PCR yield plateaus when primers deplete, as each cycle incorporates one primer per strand.

Standard 0.1–1.0 _μM concentrations confirm 0.2 _μM suits this setup, with full consumption defining theoretical maximum.

Verification Factors

Average base pair weight of 660 Da aligns with dsDNA standards (range 618–660 g/mol).

Rounding to one decimal place gives 6.6 _μg, matching precise computation.

Correct Answer: 6.6