Preparing Oligonucleotides for the Primer Collection
Eric Kofoid
12/4/00
It is important to deposit your oligonucleotides in the primer collection as soon as possible. The collection contains two tubes for each primer. One is a high concentration primary stock, usually 100 µM. The other is a standard 5 µM working stock which is used directly in many reactions and assays done in the lab.
The oligonucleotides we receive are always lyophilized and come in two forms, depending on the synthesis machine. One comes in small 1.8 mL screw-cap vials with a printed label including a concentration in "pM/µL", and the other, in large 5 mL Falcon tubes with a handwritten tape label having only the primer name.
1. Small vials
The material in these vials has been lyophilized from 0.8 mL original volume, and the concentration printed on the label refers to that original solution. To get a 100 µM concentration, add a number of µL water equal to 8 times the printed concentration. For instance, if the latter is "12.4 pM/µL", then add 99.2 µL to get a 100 µM solution.
2. Large vials
Dissolve the contents in 100 µL water. Transfer to a 1.8 mL Eppindorf vial and spin 10' in a table-top centrifuge. Transfer the supernatant to a 1.8 mL screw-cap vial. Remove 5 µL to 1 mL of water, and determine the concentration in gm/L spectrophotometrically, taking into account the 1:200 dilution. Adjust the concentration, if possible, to 100 µM.
Primer Collection
At this point, you have prepared vials for the primary stock collection. Label the cap with the TP number on a white paper or vinyl dot, and write the common name and actual concentration on the side. These vials go into boxes in the back of the freezer in room 209. The boxes are ominously labeled "DO NOT USE!", which means not for everyday consumption, let the primer tsar deal with them, etc.
Make 5 µM stocks for the working collection. When diluting a 100 µM stock, this is most easily done by removing 25 µL to 475 µL water in a 1.8 mL screw-cap vial. Label as before -- in this case the concentration will always be "5 µM". I have printed labels for the caps, if you would like to use them. These provide a nice visual reminder to the user that the vial actually belongs to the working collection and not the primary one. Boxes for these oligonucleotides are near the front of the freezer.