Difference between revisions of "Template:UPMC-Paris/Test"
| Line 427: | Line 427: | ||
<a href=""><div> | <a href=""><div> | ||
<h>Digestion</h> | <h>Digestion</h> | ||
| − | < | + | <li>1. 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: | In a 1.5mL tube combine the following: | ||
<ol> - DNA </ol> | <ol> - DNA </ol> | ||
| − | < | + | <ol>- Restriction Enzyme(s)</ol> |
| − | < | + | <ol>- Buffer </ol> |
| − | < | + | <ol>- dH2O up to total volume</ol> |
| − | <p>Mix gently by pipetting. </p></ | + | <p>Mix gently by pipetting. </p></li> |
| − | < | + | <li>2. Incubate tube at appropriate temperature (usually 37°C) for 1 hour. </li> |
| − | < | + | <li>3. Always follow the manufacturer’s instructions. </li> |
| − | < | + | <li>4. To visualize the results of your digest, conduct gel electrophoresis</li> |
</div></a> | </div></a> | ||
<a href=""><div> | <a href=""><div> | ||
| Line 445: | Line 445: | ||
<p>Combine the following in a PCR or Eppendorf tube:</p> | <p>Combine the following in a PCR or Eppendorf tube:</p> | ||
<ol>- 25ng Vector DNA</ol> | <ol>- 25ng Vector DNA</ol> | ||
| − | < | + | <ol>- 75ng Insert DNA</ol> |
| − | < | + | <ol>- Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)</ol> |
| − | < | + | <ol>- 0.5-1μL T4 DNA Ligase</ol> |
| − | < | + | <ol>- H20 to a total of 10μL</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></a></li></ | + | </div></a></li></ol></li> |
Revision as of 14:54, 29 July 2016
- Transformation :
-
Preparation - 1. Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath
- 2. Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently
- 3. In a sterile test tube add competent cell (0.2~0.5 ml) to the DNA solution (<0.1 ml) and incubate in a roller drum at 37.
- 4. Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.
Digestion - 1. 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.
- 2. Incubate tube at appropriate temperature (usually 37°C) for 1 hour.
- 3. Always follow the manufacturer’s instructions.
- 4. 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).
- 1. 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:
-
- Deletion :
- Other :
