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<h3>Digestion :</h3> | <h3>Digestion :</h3> |
Revision as of 03:20, 18 October 2016
Competentiation and transformation of E. coli
Vector modifications
Digestion :
Select restriction enzymes to digest your DNA. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
In a 1.5mL tube combine the following:
- ➟ DNA
- ➟ Restriction Enzyme(s)
- ➟ Buffer
- ➟ dH2O up to total volume
Mix gently by pipetting.
Incubate tube at appropriate temperature (usually 37°C) for 1 hour.
Always follow the manufacturer’s instructions.
To visualize the results of your digest, conduct gel electrophoresis
Vector Preparation :
Combine the following in a PCR or Eppendorf tube:
- ➟ Vector DNA
- ➟ Insert DNA
- ➟ Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)
- ➟ 0.5-1μL T4 DNA Ligase
- ➟ H20 to a total of 10μL
- To dertermine the amount of DNA needed, we used the NEB calculator
Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).