Hints and Notes for the PCR Box Titration Calculator

This JavaScript program calculates the amounts of various ingredients needed to do two-dimensional box titrations of various reactants in polmerase chain reactions. The default values are from the AmpliGrease hot-start protocol of Horton et al.⁽¹⁾ A brief description of this program has been published.⁽²⁾

Using the Program

1. Using the radio buttons, select which ingredients to put in the top mix, which to put in the bottom mix, and which to split proportionately between them.
2. Use the checkboxes to select one ingredient to titrate in the top mix, and one to titrate in the bottom mix.
3. Enter the number of titration points for the bottom mix in the columns box and the number of titration points for the top mix in the Rows box.
4. Enter stock concentrations for each ingredient.
5. Enter the final concentration for each ingredient in the far right column. This is the concentration in the reaction after top and bottom mixes are mixed.
6. The Fudge Factor adds an extra bit to account for potential pipetting errors, so you don't run out of mix before the last tube. The more accurate your pipetting, the smaller the fudge factor you can use.
7. When your values are entered, click Done. You will be asked for the final concentrations of the other titration points, then a worksheet will be calculated that has the reagent volumes needed for each step in your experiment.

To leave an ingredient out of the reaction, enter a final concentration of 0. Note that the stock concentration for each ingredient must be greater than zero, whether you use that ingredient or not. For ingredients that will be titrated, enter the highest concentration you will use.

This worksheet can be printed for your records, and/or save

d to your hard drive (using File: Save As...: source) from your browser menu.

There are many possible variations of the box titratrion that can be calculated with this program. Whether they make sense technically is up to you.

To set up multiple parallel reactions to titrate magnesium chloride for various templates, tell the program to titrate magnesium versus template, then just enter the same template concentration for each point.

References:

1. Horton, R.M., Hoppe, B.L., and Conti-Tronconi, B.M. AmpliGrease: Hot-start PCR using petroleum jelly. BioTechniques 16(1):42-43, 1994.
2. Horton, R.M., and Stone, R.J.S. PCR master mix volume calculators in JavaScript. BioTechniques 24(3), 1998