About Primers

Eric Kofoid (current primer custodian) 5/12/00

1. Always order your primers through the primer custodian.

This way they get into the database, receive TP numbers and are made available to everybody in the lab. Use email . Give each primer a pet name, include comments (what it's for, where it binds, etc), and, of course, don't forget the sequence!

2. Primers are delivered to the cold room, 2nd floor.

3. Primer preparation.

The primer custodian will be happy to do this for you. If you want to do it yourself, here are the rules:

i) OP purified primers (small vials with printed labels)

These have a concentration printed on the side (e.g., "pM/µL: 20.5"). This refers to the concentration at an original volume of 0.8 mL, before lyophilization.

To prepare a 100 µM stock, multiply the number by 8 (e.g., 20.5 x 8 = 164), and add this number of µL of Barnstadt water (or other ultrapure water).

Cross out the number, and write "100 µM" in its place.

ii) Other primers (larger 3.5 mL test-tubes with hard-to-remove caps)

Add 100 µL ultrapure water to the tube and vortex. Transfer the liquid to a 1.5 mL Eppindorf vial, spin 10' full-speed in a table-top centrifuge. Transfer the supernatant to a screw-cap 1.5 mL tube -- see the custodian if you need some.

Dilute 5 µL into 1 mL Barnstadt water. Measure the concentration using the Beckman spectrophotometer in Rm. 208. Turn the UV light on 20' before starting, use the "DNA/Oligo Quant: ss DNA" program with "background correction: YES" and "Dilution Factor: 200". The concentration given will be in µg/mL.

Convert to µM by calculating the MW, assuming 330 Daltons per average base. Divide the mass concentration (µg/mL) by this number and multiply by 1000 to get the micromolar concentration (µM). If this is over 100, dilute to give a 100 µM primary stock. Write the concentration clearly on the side of the tube.

3. Primer storage.

Put a label with the TP number on the cap, and write the pet name (e.g., "pickle22") on the side. Give the stock vial to the custodian, and he or she will freeze it with the rest of the collection.

Last Update: Thursday, 19-Jun-2014 11:50:11 PDT
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Eric Kofoid eckofoid at ucdavis.edu