Difference between revisions of "Annealing Oligos for Cloning"

Revision as of 17:29, 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

(X ng vector)/(.650 ng/pmol x Y base pairs)
Example: (10 ng)/(.65 x 2079 bp) = .0074 pmol

Calculate how many pmol of annealed oligos for the ligation

X pmol vector x (molar excess of oligos)
Example: .0074 x 6 = .0444 pmol/ul oligos

Calculate how much to dilute annealed oligos

Annealed oligos are at 5 uM, or 5 pmol/ul
Dilution factor for oligos = (5 pmol/ul)/(__ pmol/ul annealed oligos)
Example: (5 pmol/ul)/(.0444 pmol/ul) = 113

Dilute annealed oligos with dH20

Example: 1 ul annealed oligos plus 113 ul dH20

Use 1 ul of diluted oligos for ligation