Resuspending Primers

Overview

Primers are often shipped and received dry or in a lyophilized state. First create a master 100uM stock solution (1 for each primer) and then dilute into 10uM or 5uM working stock or aliquot. This reduces the number of freeze/thaw cycles the master primer stock goes through and reduces the chances of contaminating the primary source for the primer.

Procedure

1. Locate your primers in the package and ensure they are the primers you ordered.

2. Compare the nucleotide sequences and GC% on each primer label to the order invoice. The melting temperature will likely differ between the label and the order (this is not important).

3. Spin down the tubes using the centrifuge machine. SPIN ALL TUBES BEFORE OPENING! This is because dry pellet can often come dislodged during shipping and could be in the cap.

4. Create a 100uM master stock by adding MBG water into the tube. Take the amount of nanomoles (nmols) on the label and multiply it by 10. That number is how many microliters of MBG water you'll add to the tube.

Rev2 = 27.4 nmol = 274 uL MBG water

For7 = 26.6 nmol = 266 uL MBG water

5. Label the top of each primer tube with its name and concentration (100uM).

Labeled

KCNJ6 FOR7 100 uM & KCNJ6 REV2 100 uM

6. Wait 10 minutes to allow the master stock to rest at room temperature and then mix well (centrifuge) before creating the working solutions.

7. Dilute the primer master stock in a sterile 0.6mL microcentrifuge tube. Add 1uL of master stock and 19uL of MBG water into the microcentrifuge tube. This makes 20uL of a 5uM solution. OR add 2uL of master stock and 18uL of MBG Water into the micocentrifuge tube to make 20uL of 10uM solution. If the primer is a sequencing primer dilute to 5uM. If it is a mutation or subcloning primer dilute to 10uM.

8. Label the aliquot tube with the primer name and concentration. Update the online corresponding Freezer C Box document.