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 然 concentration (that's micromolar = 痠ol/L = pmol/痞). 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 然 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 然 low-concentration working solution, take 10 ul stock solution and add to 90 ul water (or some multiple thereof, like 30 痞 of high concentration stock + 270 痞 water).

General Notes

  • If you choose to resuspend your primers to a concentration other than 100 然, please write this clearly on the primer tube. If you are diluting from someone else's suspended primers, it wouldn't hurt to verify with them that the concentration is 100 然 before diluting. If you have a 200然 concentration, use 5無 primer in 95無 water to get a 10然 low-concentration working solution.
  • Sometimes micromolar (痠ol/L) concentrations are abbreviated in brackets, e.g. [100] mean 100 micromolar.
  • PCR: To use in a typical 50 無 PCR reaction, add 15 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 痞 of your low concentration [10] stock is appropriate for a 12 痞 sequencing reaction at the ICMB core. This volume gives 10 pmol of primer.
Edit | Attach | Watch | Print version | History: r9 < r8 < r7 < r6 < r5 | Backlinks | Raw View | More topic actions

 Barrick Lab  >  ProtocolList  >  ProtocolsResuspendingPrimers

Contributors to this topic Edit topic MichaelHammerling, JeffreyBarrick, JuliePerreau, LindseyWolf, AurkoDasgupta
Topic revision: r9 - 2017-01-11 - 21:31:18 - Main.JuliePerreau
This site is powered by the TWiki collaboration platform Powered by Perl This site is powered by the TWiki collaboration platformCopyright ©2024 Barrick Lab contributing authors. Ideas, requests, problems? Send feedback