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 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 reaction (using invitrogen ligase)

5x T4 DNA Ligase - 4uL Vetor – 42 ng Inserto – 100 ng T4 DNA Ligase – 1 uL Vol final - 20ul