"Digestion and restriction map"
Practical objectives:PCR
Performing digestion with a restriction enzyme.
DNA deposits in the wells of an agarose gel.
Start of electrophoresis and staining of the gel.

Theoretical objectives:
Going further into the principle of DNA electrophoresis.
Interpretation of the effect of dilution and digestion on salmon DNA.
Creation of a restriction map from the bands obtained after electrophoresis of lambda phage DNA digested by 2 restriction enzymes with the aid of instructions to establish the size of the fragments in base pairs.

Composition :
To make 4 gels of 25ml

• 100ml of TAE 10 buffer x pH =8.3 to dilute 10 times to get 1,000ml of buffer
  
• 1.6 g of agarose (for 8 gels of 25ml)
  
• 25 Pasteur pipettes calibrated for deposits in wells
  
• 0.25ml of deposit blue
  
• 0.05g of freeze-dried salmon DNA
  
• 50μl EcoR1 restriction enzyme
  
• 125μl of a DNA solution of phage Lambda digested with
  EcoR1
  
• 125μl of a DNA solution of phage Lambda digested with HindIII
  
• 125μl of a DNA solution of phage Lambda digested with EcoR1 +HindIII
  
• 10ml of sterile water
  Technical and educational instructions available on our website.
  
  Necessary material:
  
• Standard DNA electropheris tank (photos 1) :
  for versions stained a posteriori with Azure A;
  Generator 70-110 V, 20 mA
  
• BlueGel tank (photos 2) : for immediate disclosure versions without additional handling
  or standard tank + Transilluminateur
  
• Water bath or microwave
• Gloves
  
• Micropipettes from 5 to 50 µl and adpated cones
  
  Conservation: 3 months
  Enzyme and DNA: in the freezer
  Colour (protected from light), TAE buffer, sterile water: in the refrigerator