Difference between revisions of "Annealing Oligos for Cloning"
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# Turn off heat and let cool at least 2 hours | # Turn off heat and let cool at least 2 hours | ||
Revision as of 14:13, 27 June 2012
Anneal Oligos
- Design oligos (the Oligator is a useful tool for this)
- Order oligos at 100 uM concentration (eg. IDT)
- 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
- Boil 4 minutes in beaker with 400 ml H2O
- 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: (10 ng)/(670 x 2079 bp x 10 exp-6) = 7.2 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)
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