Difference between revisions of "Annealing Oligos for Cloning"
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(Created page with ' A. Reaction Components: 2 ul 10X PCR buffer w/ 15 mM MgCl2 # Design oligos (the Oligator is a useful tool for this) # Order oligos at 100 uM concentration (eg. IDT) # ul …') |
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− | + | '''Anneal Oligos''' | |
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# Design oligos (the Oligator is a useful tool for this) | # Design oligos (the Oligator is a useful tool for this) | ||
# Order oligos at 100 uM concentration (eg. IDT) | # Order oligos at 100 uM concentration (eg. IDT) | ||
− | # | + | # Set up annealing reaction |
− | ul | + | |
− | + | 2 ul 10X annealing buffer (1 M NaCl, 100 mM Tris-HCl pH 7.4) | |
− | 1 ul | + | 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 | |
− | 1 | + | Example: 1 ul annealed oligos plus 113 ul dH20 |
− | + | Use 1 ul of diluted oligos for ligation |
Revision as of 14:16, 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