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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  A. Reaction Components:
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'''Anneal Oligos'''
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2 ul 10X PCR buffer w/ 15 mM MgCl2
 
  
 
# 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)
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#  
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# Set up annealing reaction
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  ul 2 mM dNTPs (working concentration 2 mM each)  
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3
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  2 ul 10X annealing buffer (1 M NaCl, 100 mM Tris-HCl pH 7.4)
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1 ul forward primer (20 pmol)
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1 ul each oligo (5 uM final concentration)
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dH2O to 20 ul total
  
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1 ul reverse primer (20 pmol)
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# Boil 10 minutes in beaker with 400 ml H2O
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# Turn off heat and let cool at least 2 hours
  
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1 ul Taq DNA polymerase
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'''Dilute Oligos for Ligation'''
  
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12 ul dH2O
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Calculate how much to dilute annealed oligos
  
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19 ul total
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Concentration (ng/ul) = (5 X 10-6 umol/ul)(650,000 ng/umol bp)(number of base pairs)
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Example:  (5 X 10-6 umol/ul)(650,000 ng/umol bp)(20 base pairs) = 65 ng/ul
  
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add 1 ul template DNA
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Calculate concentration of annealed oligos needed for ligation
  
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Create master mix if desired
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Example:  (10 ng vector)(20 bp oligo insert/2079 bp vector)(6 molar ratio) = 0.57 ng/ul
  
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  B. Thermal Profile
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Calculate annealed oligo dilution
  
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1 circle of : 94 C 4 mins
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Example: (65 ng/ul)/(0.57 ng/ul) = 114
  
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30 circles of:  94 C 15 sec.; annealing temp. 15 sec.; 74 C 30 sec.
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Dilute annealed oligos with dH20
  
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1 circle of : 74 C 4 mins
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Example: 1 ul annealed oligos plus 113 ul dH20
  
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annealing temp. should be about 4 C below the lower of the Tm values for the two primers.
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Use 1 ul of diluted oligos for ligation

Revision as of 14:16, 1 October 2011

Anneal Oligos

  1. Design oligos (the Oligator is a useful tool for this)
  2. Order oligos at 100 uM concentration (eg. IDT)
  3. 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
  1. Boil 10 minutes in beaker with 400 ml H2O
  2. 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

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