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Protocol

Double Digestion |Gel Extraction|Miniprep | Chemical Inducible Expression of GFP |DNA Purification |Ligation of Insert DNA | PCR |Preparation of Competent Cells| Site-directed Mutagenesis| Transformation|

Protocol for Ligation of Insert DNA into Plasmid Vector DNA

</html> <a href="https://static.igem.org/mediawiki/2010/0/04/Protocol_for_ligation_of_insert_DNA_into.pdf"target="blank"><img src="" width=20>download PDF version</a> ==Materials:== Materials: * DNA sample(s) in water or TE buffer *10x ligation buffer *T4 DNA Ligase, 5 u/μl *ddwater ==Procedure:== 1. Test the concentration of the DNA sample(s). 2. Pipet the following into a microfuge tube: a. Linearized vector DNA: around 100ng b. Insert DNA (at 3:1 molar excess over vector): variable c. 10x ligation buffer: 1uL d. T4 DNA Ligase: 1uL e. ddwater: Rest of volume '''Total volume: 10 uL''' 3. Vortex and spin briefly to collect drops. 4. Incubate the mixture at 16 degree for 45-60min. 5. Use the ligation mixture for transformation. ==Tips:== * Thoroughly mix the 10x ligation buffer before use. * The optima l insert/vector molar ratio is 3:1. * To minimize recircularization of the cloning vector, dephosphorylate linearized plasmid DNA with Alkaline Phospha tase(CIAP) prior to ligation. Heats inactivate the phosphatase or remove from the mixture after the dephosphorylation step. * DNA purity is an important factor for successful ligation. Plasmids should be purified using a method that will ensure isolation of high quality DNA. Use only high quality agarose and fresh electrophoresis buffers for gel-purification of DNA fragments.