Difference between revisions of "Annealing Oligos for Cloning"

Revision as of 14:51, 21 January 2012

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 the molarity of the vector that will be in the ligation

(__ ng vector)(10 exp6)/(670 g/pmol x __ base pairs) = ___ nM

Example: (10 ng)(10 exp6/(670 x 2079 bp) = 7.2 nM

Calculate the molarity of annealed oligos to be used for the ligation

__ nM vector x (molar excess of oligos)

Example: 7.2 nM x 3 = 21.6 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

Example: 5000 nM /21.6 nM = 231 dilution factor

Dilute annealed oligos with dH20

Example: 1 ul annealed oligos plus 230 ul dH20

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