Primers

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