Difference between revisions of "Annealing Oligos for Cloning"
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dH2O to 20 ul total | dH2O to 20 ul total | ||
− | # Boil | + | # Boil 4 minutes in beaker with 400 ml H<sub>2</sub>O |
# Turn off heat and let cool at least 2 hours | # Turn off heat and let cool at least 2 hours | ||
'''Dilute Oligos for Ligation''' | '''Dilute Oligos for Ligation''' | ||
− | Calculate | + | 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 | ||
− | Example: | + | Ligation Example: 5000 nM /108 nM = 46 dilution factor |
+ | GGA Example: 5000 nM /36 nM = 139 dilution factor | ||
+ | |||
Dilute annealed oligos with dH20 | Dilute annealed oligos with dH20 | ||
− | Example: 1 ul annealed oligos plus | + | Ligation Example: 1 ul annealed oligos plus 45 ul dH20 |
+ | |||
+ | GGA Example: 1 ul annealed oligos plus 138 ul dH20 | ||
− | Use 1 ul of diluted oligos for ligation | + | 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