Resuspending Primers

  1. Your primers will arrive as a lyophilized film at the bottom of a cryo-tube. To use them, you must resuspend them in autoclaved dH2O.
  2. Make a high-concentration stock by resuspending the lyophilized primer to a standard 100 ÁM concentration (that's micromolar = Ámol/L = pmol/Ál). The amount of primer in nanomoles (nmol or nm) is provided by the vendor on the spec sheet and the side of the tube. For example, for 28.5 nmol, add 285 ul water to get a 100 ÁM stock solution. Pipette up and down until the film dissolves (you can watch it through the bottom of the tube) or resuspend by vortexing vigorously for 10 seconds.
  3. To prepare a 10 ÁM low-concentration working solution, take 10 ul stock solution and add to 90 ul water (or some multiple thereof, like 20 Ál of high concentration stock + 270 Ál water).

General Notes

  • Sometimes micromolar (Ámol/L) concentrations are abbreviated in brackets, e.g. [100] mean 100 micromolar.
  • PCR: To use in a typical 50 ÁL PCR reaction, add 1ľ5 uL of working solution of each primer to the reaction (final concentration from 200 pM to 1000 pM) depending on the size of your product. Adjust for reactions of different volume.
  • Sequencing: 1 Ál of your low concentration [10] stock is appropriate for a 12 Ál sequencing reaction at the ICMB core. This volume gives 10 pmol of primer.
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Topic revision: r5 - 09 Jun 2011 - 20:44:53 - Main.JeffreyBarrick
 
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