Difference between revisions of "Annealing Oligos for Cloning"

Revision as of 17:12, 11 October 2011

Anneal Oligos

1. Design oligos (the Oligator is a useful tool for this)
2. Order oligos at 100 uM concentration (eg. IDT)
3. Set up annealing reaction

2 ul 10X annealing buffer (1 M NaCl, 100 mM Tris-HCl pH 7.4)
1 ul each oligo (5 uM final concentration)
dH2O to 20 ul total

1. Boil 10 minutes in beaker with 400 ml H2O
2. Turn off heat and let cool at least 2 hours

Dilute Oligos for Ligation

Calculate how many pmol of vector will be in the ligation

Calculate how much to dilute annealed oligos

Concentration (ng/ul) = (5 X 10-6 umol/ul)(650,000 ng/umol bp)(number of base pairs)
Example:  (5 X 10-6 umol/ul)(650,000 ng/umol bp)(20 base pairs) = 65 ng/ul

Calculate concentration of annealed oligos needed for ligation

Example:  (10 ng vector)(20 bp oligo insert/2079 bp vector)(6 molar ratio) = 0.57 ng/ul

Calculate annealed oligo dilution

Example: (65 ng/ul)/(0.57 ng/ul) = 114 

Dilute annealed oligos with dH20

Example: 1 ul annealed oligos plus 113 ul dH20

Use 1 ul of diluted oligos for ligation