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Team:Peking S/lab/protocol/insert
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==Materials:== | ==Materials:== | ||
Materials: | Materials: | ||
Latest revision as of 08:43, 2 October 2011
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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
download PDF versionMaterials:
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.
