GATE 2023 BT – Question 65

Recombinant DNA Technology and Other Tools in Biotechnology · Molecular tools: PCR, DNA and RNA labelling, Sanger and next generation sequencing, Southern and northern blotting, in-situ hybridization, DNA fingerprinting · 2 marks · Numerical answer

A dNTP master mix combines 40 μL of each 20 mM dATP, dCTP, dGTP and dTTP stock. 4 μL of this mix is added to a PCR mix of final volume 50 μL. The concentration of total dNTPs (μM) is __________.

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Correct answer: 1600

Explanation

**Step 1: concentration of each dNTP in the master mix.** The master mix is made from 40 μL of each of the four 20 mM stocks, so the total volume is $4\times40=160$ μL. Each nucleotide is diluted four-fold:
$$\frac{20\times40}{160}=5\text{ mM each}.$$

**Step 2: total dNTP in the master mix.**
$$4\times5\text{ mM}=20\text{ mM}.$$

**Step 3: dilute into the PCR.** 4 μL of the master mix goes into a final volume of 50 μL:
$$C_{final}=20\text{ mM}\times\frac{4}{50}=1.6\text{ mM}=\mathbf{1600\ \mu M}$$
(about 400 μM of each of the four dNTPs).