Difference between revisions of "Template:UPMC-Paris/Test"
| Line 428: | Line 428: | ||
<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. | <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:</li> |
<ol> - DNA </ol> | <ol> - DNA </ol> | ||
<ol>- Restriction Enzyme(s)</ol> | <ol>- Restriction Enzyme(s)</ol> | ||
<ol>- Buffer </ol> | <ol>- Buffer </ol> | ||
<ol>- dH2O up to total volume</ol> | <ol>- dH2O up to total volume</ol> | ||
| − | <p>Mix gently by pipetting. </p | + | <p>Mix gently by pipetting. </p> |
<li>2. Incubate tube at appropriate temperature (usually 37°C) for 1 hour. </li> | <li>2. Incubate tube at appropriate temperature (usually 37°C) for 1 hour. </li> | ||
Revision as of 14:55, 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).
-
- Deletion :
- Other :
