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<p>Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.</p> | <p>Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.</p> | ||
− | <div class="image1" id="TransfoPic1"/> | + | <div class="image1" id="TransfoPic1"/></div> |
<h3>Digestion :</h3> | <h3>Digestion :</h3> | ||
<p>Select restriction enzymes to digest your plasmid. Determine an appropriate reaction buffer by reading the instructions for your enzyme. </p> | <p>Select restriction enzymes to digest your plasmid. Determine an appropriate reaction buffer by reading the instructions for your enzyme. </p> |
Revision as of 13:06, 14 October 2016
Competent Cells
Transformation
Digestion :
Select restriction enzymes to digest your plasmid. 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:
- -> 25ng Vector DNA
- -> 75ng 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
Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).