Barrick Lab :: ProtocolsResuspendingPrimers

---+ Resuspending Primers

   1 Your primers will arrive as a [[http://en.wikipedia.org/wiki/Freeze-drying][lyophilized]] film at the bottom of a cryo-tube.  To use them, you must resuspend them in autoclaved dH<sub>2</sub>O.
   1 Make a *high-concentration* stock by resuspending the lyophilized primer to a standard 100 µM concentration (that's micromolar = µmol/L = pmol/µl). 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 µM 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.
   1 To prepare a 10 µM *low-concentration* working solution, take 10 ul stock solution and add to 90 ul water (or some multiple thereof, like 30 µl of high concentration stock + 270 µl water).

*General Notes*
   * If you choose to resuspend your primers to a concentration other than 100 µM, 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 µM before diluting. If you have a 200µM concentration, use 5µL primer in 95µL water to get a 10µM low-concentration working solution.
   * Sometimes micromolar (µmol/L) concentrations are abbreviated in brackets, e.g. [100] mean 100 micromolar.
   * PCR: To use in a typical 50 µL PCR reaction, add 1–5 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 µl of your low concentration [10] stock is appropriate for a 12 µl sequencing reaction at the ICMB core. This volume gives 10 pmol of primer.

Barrick Lab > ProtocolList > ProtocolsResuspendingPrimers

Contributors to this topic

MichaelHammerling, JeffreyBarrick, JuliePerreau, LindseyWolf, AurkoDasgupta

Topic revision: r9 - 2017-01-11 - 21:31:18 - Main.JuliePerreau