OLIGONUCLEOTIDES and dilution of primers for PCR

 

1. Currently primers are ordered from SIGMA GENOSYS or others depending on price (remember to consider delivery charges and difference costs with redundancies).

2. Use the website order system that contains correct information about scales available - if you are ordering more than c. 6 primers, the bulk submission is simpler.

3. Order small scale: 0.05 um or (less) 3 OD (but note from Sigma-Genosys that the minimum scale is the same price as the next size up so order the larger; sometimes the minimum scale is not available for oligos containing redundancies or modifications).

4. When the oligos come, put the original datasheet and gel picture in the Oligo Master Book. Make a photocopy for yourself.

5. In the laminar flow hood, reconstitute the dried oligos in (e.g. SIGMA) molecular biology grade water to make a 100 M (micro-molar) stock solution; in addition to the original stock tube make two 20l (micro-litre) stock aliquots. (Oligos will be more stable in slightly buffered salts, e.g. TE, but this can inhibit enzyme reactions.)   The data sheet will give the number of ul of water that are needed to make this dilution - typically 300 to 800 µl. (They also give the nanomoles in the synthesis - e.g. 46.6 nmole. To make the 100 uM stock, multiply this by 10 and add than many ul of water (e.g. 233 µl for 46.6 nmol = 200 µM stock solution).

6. Write the name of the oligo on the cap of the stock tube. Then put the original stock in a common oligo box (normally large fluorescent boxes in tall freezers in Trude's or Pat's drawer).

7. Small aliquots of specific oligo stocks can be kept in individual boxes in your freezer compartment..

8. Make a small amount of working solution by diluting aliquoted 100 µM stock (in the laminar flow hood) with molecular biology grade water.

- 1:10 giving a 10 M solution for genomic PCR If you use 1 µl of this stock in a 20 µl PCR mix, your are using 0.4 µM of primer in the final mix.

- 1:20 giving a 5M for plasmid PCR

9. PCR set-up using YorkBio or Promega Taq polymerase (formerly we used Bioline, now more expensive):

· water (add first)

· 1.5 1 of 50mM MgCl (final conc. 1.5 mM)

· 5 l PCR buffer (comes with Taq)

· 1 l 10 mM dNTPmix or 41 of 2.5 mM dNTPmix (final conc. 200 M; see below)

· 2.5 l of each primer'

· 5-10 ng of DNA

· 0.1-0.5 l Taq polymerase (=1 unit to 5 units)

· TOTAL 50l

 Notes

· Scaling down of PCR set-up to 20-30l for marker typing and testing (even as low as 7l is possible). Only use 50 µl wher you need to cut out and clone the

· dNTP mix: we used to make a 2.5mM working solution in Tris-HCI but ROCHE recommends to make a 10mM solution by adding 51 of dATP, dCTP, TTP and dGTC (each 100mM; as supplied by ROCHE) to 30l sterile double distilled water.

 BE AWARE THAT DILUTED OLIGOS CAN BREAK DOWN WHEN DEFROSTED SEVERAL TIMES.

 ALWAYS WEAR GLOVES WHEN HANDLING OLIGOS.

 M13 primer STOCK AND DILUTED ALIQUOTS WILL BE KEPT IN THE GENERAL PCR BOX.

Trude Schwarzacher

January 2004 / PHH Sept. 2008