Michael Court
Updated Dec 2002
PCR, Sequencing and QPCR primer preparation
1.
Important!!! – always use fresh PCR water to reduce risk of cross contamination of
new primers.
2.
Newly synthesized PCR primers (“oligos”) are provided dried down with an ODU
reading (from UV absorbance at 260 nm) which indicates how much DNA is in the tube.
a. 1 ODU = 33 ug of oligo
b. e.g. 5 ODU = 165 ug oligo
3.
Make up a “stock” concentration solution:
a. Add PCR water to dissolve and a make “stock” concentration solution of 50 uM (50
pmoles/uL) using the following formula:
# uL PCR water to add to tube = (# ODU x 2,000) / # bases in primer
b. For example: 5 ODU of a 25 base primer ----- Add 5 x 2,000 / 25 = 400 uL to tube
c. Label tube with primer number and concentration
d. Store all “stock” concentration solutions of primers in the primer boxes (labeled
primer 1, 2, etc) in the -800C freezer
4.
Make up a “working” concentration solution
a. Dilute some of the 50 uM solution 10 times to 5 uM (5 pmoles / uL) “working”
concentration with PCR water
b. For example: Add 50 ul “Stock solution” plus 450 uL of PCR water
c. Label tube with primer number and concentration
d. Store these in the -200C freezer