^*Master mixes contain an extra " + document.pcr.fudge.value + "% to allow for pipetting errors. These values are rounded to the nearest tenth of a microliter."); PrWin.document.writeln(

This program is patterned loosely after the Macro for Microsoft Excel described in reference 1 of the Notes. "); HelpWin.document.writeln("Enter the variables for your experiment, then press Calculate to have the program determine the amounts of each reagent to use."); HelpWin.document.writeln("The percent extra for pipetting errors is to insure that you don't run out of master mix before you finish all of your reactions. "); HelpWin.document.writeln("The more accurate your pipetting,the smaller this amount needs to be."); HelpWin.document.writeln("

When your variables are entered and the volumes have been calculated, click Print to have the program generate a worksheet for setting up your reaction. "); HelpWin.document.writeln("You can print a copy of this worksheet for your records."); HelpWin.document.writeln("

While some of the reagent concentrations can be changed by entering new values in the text boxes,others are built into the program."); HelpWin.document.writeln("These default values are given below. They can be changed fairly easily by editing the program."); HelpWin.document.writeln("

To change these defaults, you must edit the program. Follow these simple steps:"); HelpWin.document.writeln("

"); HelpWin.document.writeln("
1. Save your own copy of the program using your browser's File:Save As option. Be sure to save it as Source."); HelpWin.document.writeln("
2. Open the saved file in a text editor or word processor."); HelpWin.document.writeln("
3. Find the setDefaults function at the top of the program."); HelpWin.document.writeln("
4. Change the numbers to use your own defaults."); HelpWin.document.writeln("
5. Save the file. If you are using a word processor, be sure to save it as text."); HelpWin.document.writeln("
6. Open your modified version of the file in your browser. For netscape this is done from the top menu with File:Open:Page in Navigator. It should now have your defaults. Check this Notes screen to be sure."); HelpWin.document.writeln("

The original default values are given below. "); HelpWin.document.writeln("Note that the values in the table above will change if you edit the default values, but the example below will not. "); HelpWin.document.writeln("This gives you a record of what the defaults were originally. "); HelpWin.document.writeln("

function setDefaults() {");
  HelpWin.document.writeln("	// set default reaction volumes (volumes in microliters per reaction). ");
  HelpWin.document.writeln("	// Change the numbers here to use different defaults.");
  HelpWin.document.writeln("	");
  HelpWin.document.writeln("	ulStandardRxnVol = 25;  // Standard reaction volume");
  HelpWin.document.writeln("	ulPrimer1PerRxn = 1;    // microliters of primer 1 used per standard reaction");
  HelpWin.document.writeln("	ulPrimer2PerRxn = 1;    // microliters of primer 2 used per standard reaction");
  HelpWin.document.writeln("	ulBufferPerRxn = 2.5;   // microliters of buffer stock used per standard reaction");
  HelpWin.document.writeln("	uldNTPPerRxn = 2;       // microliters of dNTP stock used per standard reaction");
  HelpWin.document.writeln("	ulTaqPerRxn = 1;        // microliters of polymerase used per standard reaction");
  HelpWin.document.writeln("	");
  HelpWin.document.writeln("	document.pcr.RMvol.value  = ulStandardRxnVol // Standard reaction volume");
  HelpWin.document.writeln("	document.pcr.Ntubes.value = 10;    // number of reaction tubes");
  HelpWin.document.writeln("	document.pcr.Tvol.value = 2;       // template volume per standard reaction");
  HelpWin.document.writeln("	document.pcr.fudge.value = 10.0    // percent extra for pipetting errors");
  HelpWin.document.writeln("	document.pcr.dmsoconc.value = 0    // DMSO concentration (use 0 to omit DMSO)");
  HelpWin.document.writeln("	document.pcr.mgclconc.value = 1.5  // final concentration of MgCl2 in reaction, millimolar");
  HelpWin.document.writeln("	document.pcr.mgclstk.value = 25    // stock concentration of MgCl2, millimolar");
  HelpWin.document.writeln("}

"); HelpWin.document.writeln("
• Polymerase concentration: 0.25 to 0.5 Units/ml
  "); HelpWin.document.writeln("
• Primer concentration: 5 to 25 mM
  "); HelpWin.document.writeln("
• dNTP concentrations: 0.5 to 2.5 mM each
  "); HelpWin.document.writeln("

References:

"); NoteWin.document.writeln("
1. Stowe, R.P. and Pierson, D.L. Spreadsheet Macro for Setting up PCR Assay Tubes. BioTechniques 20(6):1088-1089, 1996."); NoteWin.document.writeln("
2. Horton, R.M. and Stone, R.J.S. BioTechniques (in press)"); NoteWin.document.writeln("

"); NoteWin.document.writeln("

Related Calculators:

"); NoteWin.document.writeln("
• PCR Box Titration Calculator"); NoteWin.document.writeln("

Web Sites on PCR:

"); NoteWin.document.writeln("
• PCR: DNA Amplification Protocol
  "); NoteWin.document.writeln("
• Polymerase Chain Reaction Protocol p4
  "); NoteWin.document.writeln("
• DNA Amplification Protocol Main Page
  "); NoteWin.document.writeln("
• PCR
  "); NoteWin.document.writeln("
• Targeted PCR Using Pine Genomic Templates
  "); NoteWin.document.writeln("
• TJL IMR: Optimizing PCR
  "); NoteWin.document.writeln("
• Standard PCR
  "); NoteWin.document.writeln("