Sunday, September 7, 2008

Preparation of Stock PCR Primers

Always take great care not to contaminate the original primer stock - use filter tips and PCR-quality reagents

1. When you receive the lyophilized primer, before opening it be sure to spin it down to insure that the primer pellet is at the bottom of the tube.

2. Prepare the 100 uM primer stock solution in the tube containing the pellet. This tube will be used to make working primer solutions as needed and will be stored at -20°C or below.

To prepare stock:

Determine how many nmoles of primer is in the tube (usually written on the tube or on information accompanying primer order).

Resuspend the pellet in CLEAN 10mM Tris, pH 8.8 buffer to generate the 100uM stock solution. An easy way to do this is to multiply the nmole value by 10 and add that volume in ul to the pellet.

Example: If primer pellet is 8.3 nm, you will add 83 ul 10mM Tris, pH 8.8 buffer to the pellet to resuspend it. The final concentration of primer is 100 uM.

3. To generate the 20 uM working dilution of the primer, dilute the stock solution 1:5 with 10mM Tris, pH 8.8. Example: 10ul stock + 40ul 10mM Tris buffer.