OLIGONUCLEOTIDES
and dilution of primers for PCR – www.molcyt.com |
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 µM (micro-molar) stock
solution; in addition to the original stock tube make two 20µl (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 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
µM 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 5µM
for plasmid PCR 9. PCR set-up using YorkBio or · water (add first) · 1.5 µ1 of 50mM MgCl (final conc. 1.5 mM) · 5 µl PCR buffer
(comes with Taq) · 1 µl 10 mM dNTPmix or 4µ1 of 2.5 mM dNTPmix (final conc. 200 µM;
see below) · 2.5 µl of each
primer' · 5-10 ng of DNA · 0.1-0.5 µl Taq polymerase (=1 unit to 5 units) · TOTAL 50µl Notes · Scaling down of
PCR set-up to 20-30µl for marker typing and testing (even as low as 7µl 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 5µ1 of dATP, dCTP,
TTP and dGTC (each 100mM; as supplied by ROCHE) to
30µl 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. January 2004 / PHH
Sept. 2008 – from www.molcyt.com or www.molecularcytogenetics.com |