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-   -   DNA Ligations Factors Affecting Efficiency (http://www.molecularstation.com/forum/dna-techniques/2037-dna-ligations-factors-affecting-efficiency.html)

kiki06 09-17-2007 07:20 AM

DNA Ligations Factors Affecting Efficiency
 
Hello guys,

Factors affecting DNA Ligation efficiency

Optimal Ligation Effiency was using:
  • 50 ng of vector
  • Overnight Ligation
  • 16°C
  • 2:1 insert:vector molar ratio
  • standard T4 ligase used
  • Ligase was heat inactivated at 65°C for 20 mins
  • 2 μL / 20 μL was used to transform commercial heat-shock competent cells.
(7.2 kb vector + 0.6 kb insert with both sticky ends).



Now What affects Ligation Efficiency:


DNA ligation efficiency was marginally decreased by


  • Using 100 ng vector (more DNA is not better!)
  • Doing a 1 hr ligation at room temperature
  • Using insert:vector molar ratios of 5:1 and 1:1



Ligation efficiency was noticably decreased (x100) by
  • Sticky end ligation with a larger insert (5.2 kb vector + 2.6 kb insert)
  • Blunt end ligation
Ligation efficiency was severely decreased (x10000) by
  • Using DNA fragments that have been exposed to ethidium bromide and UV during the gel extraction procedure (difficult to avoid but heartily recommended)
  • Using the NEB Quick Ligation Kit (possibly a bad batch)
If anyone can post more ideas here that would be great :wub:

Kiki


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