Cloning of DNA fragments: ligation reactions in agarose gel.
Ligation reactions to ligate a desired DNA fragment into a vector can be challenging to beginners and especially if the amount of the insert is limiting. Although additives known as crowding agents, such as PEG 8000, added to the ligation mixes can increase the success one has with ligation reactions, in practice the amount of insert used in the ligation can determine the success or the failure of the ligation reaction. The method described here, which uses insert DNA in gel slice added directly into the ligation reaction, has two benefits: (a) using agarose as the crowding agent and (b) reducing steps of insert purification. The use of rapid ligation buffer and incubation of the ligation reaction at room temperature greatly increase the efficiency of the ligation reaction even for blunt-ended ligation.
Centre for Nutrition and Food Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD, Australia.
Agnelo
A
Furtado
D003001:Cloning, Molecular / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods