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Gradient PCR Planner

Plan a gradient PCR: enter the lowest and highest temperatures and the number of columns to list the temperature each column will reach.

Formula

ΔT=Thigh−Tlowncolumns−1\Delta T = \dfrac{T_{\text{high}} - T_{\text{low}}}{n_{\text{columns}} - 1}
Tk=Tlow+(k−1) ΔTT_k = T_{\text{low}} + (k-1)\,\Delta T
columns\text{columns}
number of independently heated columns in the block
ΔT\Delta T
temperature difference between neighbouring columns

How it works

A gradient thermal cycler holds the block at a range of temperatures across its columns during the annealing step, so one run tests many temperatures. The temperature of each column is the lowest value plus its share of the range, linearly interpolated.

Choose a range that brackets the estimates from the Rychlik formula and the lower primer Tm minus 5 °C. The column with the cleanest, strongest product of the expected size gives the annealing temperature to use.

Worked example

A 12-column gradient from 52 to 64 °C.

  1. Step = (64 − 52) / 11 = 1.09 °C.
  2. Column 1 is 52.0 °C, column 7 is 52 + 6 × 1.09 = 58.5 °C, column 12 is 64.0 °C.

Twelve temperatures from 52.0 to 64.0 °C, about 1.09 °C apart.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The temperature varies linearly across the columns.
  • The instrument reaches and holds each nominal temperature.

Common mistakes

  • Trusting the nominal temperatures of a block whose gradient is not linear. Check the instrument's own report.
  • Loading the samples so that the column order does not match the plan.
  • Choosing a gradient so wide that neighbouring columns differ too much to resolve the optimum.

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Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.