DNA Ligations Factors Affecting Efficiency

[kiki06]

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

Kiki