Difference between revisions of "Annealing Oligos for Cloning"
From GcatWiki
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'''Anneal Oligos''' | '''Anneal Oligos''' | ||
| − | # Design oligos (the Oligator is a useful tool for this) | + | # Design oligos (the [http://gcat.davidson.edu/igem10/ Oligator] is a useful tool for this) |
| − | # Order oligos at 100 uM concentration (eg. IDT) | + | # Order oligos at 100 uM concentration (eg. [http://www.idtdna.com/Home/Home.aspx IDT]) |
# Set up annealing reaction | # Set up annealing reaction | ||
Revision as of 14:19, 1 October 2011
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 10 minutes in beaker with 400 ml H2O
- Turn off heat and let cool at least 2 hours
Dilute Oligos for 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
