Difference between revisions of "Team:UPMC-Paris/Experiments"

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.

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

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 Step 1 :

Deletion Step 2 :

Deletion Step 3 :

Whatever Step 1 :

Whatever Step 2 :

Un autre truc Step 1 :

Un autre truc Step 2 :