IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-4

Ligation

• J36011 \SP + J36010 \XP
• J36012 \SP + J36010 \XP

For ligation, we use the Roche 5min quick ligase kit. Vials 2 and 1 are proprietary, but Vial 3 is generic T4 DNA ligase.

It is still unknown if a 1:3 volume ratio or a 1:3 molar ratio is ideal for ligations. I personally use the latter and would recommend that over the former. 1:1 may be good for small inserts, big vectors; we havent tested it though so we're not sure.

For J3011+J36010:

 1.5 uL vector DNA @65.4ng/uL
 3*(1600 /3750)* 98.1ng from above = 125.6 ng insert DNA @ 33.5 ng/uL = 3.75 uL
 2uL 5X DNA dilution buffer
 2.75uL dH20
 10ul Ligation buffer
 1ul ligase

For J3012+J36010:

 1.5 uL vector DNA @60.6ng/uL
 3*(1600 /3150)* 98.1ng from above = 149.5 ng insert DNA @ 33.5 ng/uL = 4.46 uL
 2uL 5X DNA dilution buffer
 2.04 uL dH20
 10ul Ligation buffer
 1ul ligase

• Make sure the reaction is 10uL!
• Also make sure the total amount of DNA does not exceed 200ng. Efficiency won't improve with that much DNA.
• USE the 5x DNA dilution buffer; do not skimp on it!
• Add 10uL tube 1, 1uL tube 3.
• Let sit at Room Temperature for 30min to 1h or longer.