Annealing Oligos for Cloning

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

Dilute Oligos for Ligation

Calculate the molarity of the vector that will be used in the ligation (assuming you will use 1 ul of the vector)

(__ ng vector)/(670 ng/nmol-base pair x __ base pairs x 10 exp-6 L) = ___ nM (nmol/L)

Example: (50 ng)/(670 x 2079 bp x 10 exp-6) = 36 nM

Calculate the molarity of annealed oligos to be used for the ligation (assuming your will use 1 ul of the diluted oligos)

__ nM vector x (molar excess of oligos)

Ligation Example: 36 nM x 3 = 108 nM oligos

GGA Example: 36 nM x 1 = 36 nM oligos

Calculate how much to dilute annealed oligos

Stock oligos = 100 uM
Annealed oligos = 5 uM = 5000 nM
Dilution factor for oligos = 5000 nM / desired oligo molarity

Ligation Example: 5000 nM /108 nM = 46 dilution factor

GGA Example: 5000 nM /36 nM = 139 dilution factor

Dilute annealed oligos with dH20

Ligation Example: 1 ul annealed oligos plus 45 ul dH20

GGA Example: 1 ul annealed oligos plus 138 ul dH20

Use 1 ul of vector and 1 ul of diluted oligos for ligation