Rehydrating New Primers
Follow this protocol to rehydrate lyophilized primers.
1. Place all primer vials into the centrifuge and spin at ~13k rpm for 1 min to make sure the primer is at the bottom of the tube and will not float out as you open the vial.
2. Check the label of the vial. It should contain the amount of oligo in nanomoles. To make a 100mM stock solution of your primer, add 10x the amount of nanomoles of primer in uL. So if you have 18.5 nmol of primer, add 185uL of PCR water to the tube using a sterile filter tip. This volume will change with every tube. Be extra careful here to use a new tip every time and do not cross-contaminate your primer stocks.
Note-this rehydration volume comes from the Molarity=moles/L equation. You should do the calculation by hand once so you understand why you are doing this.
3. Vortex tubes briefly and then centrifuge again at ~13k rpm for about 30 seconds just to pull everything to the bottom of the tube.
4. You now need to make the 10mM working stock of your primers that will be used for PCR.
5. Label a clean 1.5mL microfuge tube for each of the primers and label clearly which is the Fwd and which is the Rev for each gene. Make sure the full gene name is somewhere on the tube as well.
6. Add 90uL of PCR water to each tube, and then add 10uL of each primer stock solution to the corresponding tube. This creates a 1:10 dilution and gives you a 10mM working stock.
- keep on ice