Line 62: | Line 62: | ||
<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> | ||
− | <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 02:57, 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).