Line 40: | Line 40: | ||
<h3>Digestion :</h3> | <h3>Digestion :</h3> | ||
− | <p>Select restriction enzymes to digest your | + | <p>Select restriction enzymes to digest your DNA. Determine an appropriate reaction buffer by reading the instructions for your enzyme. </p> |
<p>In a 1.5mL tube combine the following:</p> | <p>In a 1.5mL tube combine the following:</p> | ||
<ol> | <ol> | ||
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>➟ Vector DNA</li> |
− | <li>➟ | + | <li>➟ 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></ol> | + | <li>➟ H20 to a total of 10μL</li> |
+ | <li>➟ To dertermine the amount of DNA needed, we used the NEB calculator</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 02:56, 18 October 2016
Competent Cells
Transformation
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).