When ordering lyophilized primers from the university or Invitrogen, they have to be resuspended in the correct amount of water so as to use the correct amount for PCR reactions.
· Add sterile ddH 2 O (using barrier tips) to resuspend the dried primer to a concentration of 100 μ M
o Each primer will come with a certificate of analysis which shows the nmoles of primer provided. To determine how much water to add, multiply the nmoles number by 10 and add that many μl of water.
o For example, if the primer provided has 44.13 nmoles, add 441.3μl of water.
o Store primer stock at -80°C
· To make a 10μM working solution of primer, dilute the 100μM stock 1:10 with sterile ddH 2 O
o For example, to make 200 μl if working primer stock, add 10μl of 100μM primer to 90μl of sterile ddH 2 O. For primers that are used often, a larger working solution can be made.
o Store working solution at -20°C
· 10μM primer solution is diluted 1:25 in a PCR reaction
o This results in a .4μM final concentration in the PCR reaction for each primer
o For example, in a 15μl PCR reaction, .6μl of each primer are added