Line 43: | Line 43: | ||
<p>In a 1.5mL tube combine the following:</p> | <p>In a 1.5mL tube combine the following:</p> | ||
<ol> | <ol> | ||
− | <li> | + | <li>➟ DNA </li> |
− | <li> | + | <li>➟ Restriction Enzyme(s)</li> |
− | <li> | + | <li>➟ Buffer </li> |
− | <li> | + | <li>➟ dH2O up to total volume</li> |
</ol> | </ol> | ||
<p>Mix gently by pipetting. </p> | <p>Mix gently by pipetting. </p> | ||
Line 57: | Line 57: | ||
<p>Combine the following in a PCR or Eppendorf tube:</p> | <p>Combine the following in a PCR or Eppendorf tube:</p> | ||
<ol> | <ol> | ||
− | <li> | + | <li>➟ 25ng Vector DNA</li> |
− | <li> | + | <li>➟ 75ng Insert DNA</li> |
− | <li> | + | <li>➟ Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)</li> |
− | <li> | + | <li>➟ 0.5-1μL T4 DNA Ligase</li> |
− | <li> | + | <li>➟ H20 to a total of 10μL</li></ol> |
<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 13:30, 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).