Molecular biology lab calculations: study guide

Key ideas

Molecular biology benches run on a small set of calculations: how much DNA is in a tube, how to dilute it, how much insert to ligate, and what temperature a primer anneals at.

• Double-stranded DNA at A₂₆₀ = 1 is about 50 µg/mL. Multiply by the dilution factor, and by 40 for RNA and 33 for single-stranded DNA.
• The Wallace rule for short primers is Tm = 2 × (A + T) + 4 × (G + C), in °C. Anneal a few degrees below the lower Tm of the pair.
• For ligation, the mass of insert = molar ratio × mass of vector × (insert length ÷ vector length).
• A serial dilution multiplies the factors, so three 1:10 steps give 1:1000. Copy number = mass × 6.022 × 10²³ ÷ (length × 660 × 10⁹), with the mass in nanograms.

Key equations

• conc (µg/mL) = A₂₆₀ × 50 × dilution
• Tm = 2(A+T) + 4(G+C)
• insert ng = ratio × vector ng × insert length ÷ vector length
• copies = mass × 6.022×10²³ ÷ (length × 660 × 10⁹)

Worked example

A 1:10 dilution of a DNA sample reads A₂₆₀ = 0.50, so the stock is 0.50 × 50 × 10 = 250 µg/mL. A 20-mer primer with 10 A or T and 10 G or C has Tm = 2 × 10 + 4 × 10 = 60 °C. For a 3 kb vector at 50 ng and a 1 kb insert at a 3:1 molar ratio, insert = 3 × 50 × (1 ÷ 3) = 50 ng.

Common mistakes

• Forgetting the dilution factor when converting an A₂₆₀ reading into a concentration.
• Using a mass ratio when a molar ratio is asked for.
• Using the Wallace rule for long primers, where salt-adjusted formulas are needed.

Practice

Test yourself with practice questions on this topic: Molecular biology lab calculations