Line 56: | Line 56: | ||
<h3>Vector Preparation :</h3> | <h3>Vector Preparation :</h3> | ||
<p>Combine the following in a PCR or Eppendorf tube:</p> | <p>Combine the following in a PCR or Eppendorf tube:</p> | ||
− | < | + | <ul> |
<li>-> 25ng Vector DNA</li> | <li>-> 25ng Vector DNA</li> | ||
<li>-> 75ng Insert DNA</li> | <li>-> 75ng Insert DNA</li> | ||
<li>-> Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)</li> | <li>-> Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)</li> | ||
<li>-> 0.5-1μL T4 DNA Ligase</li> | <li>-> 0.5-1μL T4 DNA Ligase</li> | ||
− | <li>-> H20 to a total of 10μL</li></ | + | <li>-> H20 to a total of 10μL</li></ul> |
<p>Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).</p> | <p>Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).</p> | ||
</div> | </div> |
Revision as of 11:52, 15 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).