Team:UNICAMP-EMSE Brazil/protocols/Ligation

Ligation:

• Quantificate the plasmid using absorbance at 260 nm and 280 nm.
• Concentration of plasmid (μg/mL) = A260*50*dilution
• Ratio 260/280 nm should be between 1,8-2,0.

• Use [http://www.promega.com/techserv/tools/biomath/calc06.htm this online tool] to calculate how much insert to calculate how much insert we will need to use in the reaction (http://www.promega.com/techserv/tools/biomath/calc06.htm).

Or

• [length of insert (kb)/ length of vector (kb)]* ng of vector = ng of insert for a 1:1 ratio

It´s better to use at least 100 ng of vector and a reaction ratio of 3:1 (insert:vector)

Example:

• Length of insert (in kb) = 0.7
• Amount of vector (in ng) = 100
• Length of vector (in kb) = 5

• You need 14 ng de insert for a 1:1 molar ratio
• For the 3:1= 42 ng de inserto.

Ligation (protocol adapted from Fermentas)

• Plasmid (~50-60ng)
• Insert DNA purified (the ratio was determined according to the fragment and plasmid sizes, ranging from 1:1 to 1:4)
• 2μl of T4 DNA Ligase Buffer
• 1μl of T4 DNA Ligase
• Sterile water

• Final volume = 20μl

Add the reaction components to a sterile 0,2 sterile tube. Mix gently.

Keep the tubes at 22˚C for 1 hour or overnight at 4˚C.