Difference between revisions of "Annealing Oligos for Cloning"
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| − | + | '''Anneal Oligos''' | |
| − | + | # Design oligos (the [http://gcat.davidson.edu/igem10/ Oligator] is a useful tool for this) | |
| + | # Order oligos at 100 uM concentration (eg. [http://www.idtdna.com/Home/Home.aspx 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 | |
| − | ul | ||
| − | |||
| − | |||
| − | + | # Boil 4 minutes in beaker with 400 ml H<sub>2</sub>O | |
| + | # 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 | ||
Latest revision as of 15:02, 4 July 2013
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: (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
