Team:KULeuven/Protocols

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Protocols

Transformation of E. Coli

Preparing a cell structure
At present we use TOP10F¹ cells which allow both plasmid and M13 bacteriophage transformation (For M13 transformation you need the F¹ plasmid for absorption of the bacteriophages).

Streak the TOP10F¹ cells on a tetracyclin (15 g/ml) streptomycine (20 g/ml) LB-agar plate and grow overnight
pick a single colony and grow overnight in 3 ml LB at 37C
inoculate 100 ml LB with 1 ml of the overnight culture and grow at 37C for 2-3 hours (OD₆₀₀=0.5)

CaCL₂ method

collect the bacterial cells by centrifugation (6000 rpm, 5 min, 4C)
resuspend the pellet in 40 ml 0.1 M CaCL₂ and keep on ice for at least one hour
centrifuge 5 min. at 6000 rpm, 4C
resuspend the pellet in 1 ml 0.1 M CaCL₂

use immediately or store at -70C with addition of glycerol to a final concentration of 15%
add 100 l competent cells to 20 l ice cold ligation mix in a microcentrifuge tube
keep on ice for at least 15 min.
heat shock for 25 sec. at 42C and place on ice for 2 min.
add 1 ml LB and incubate while shaking at 37C for 40 min.
plate 100 l and 900 l (spin down and resuspend in 100 l) on seperate LB plates with the required antibiotic (pGEM, pUC, all yeast shuttle vectors: 100 g/ml ampicilline)
incubate overnight at 37C

Agarose Gel Electrophoresis

Estimated time
2 hours

Materials needed

• 2 g Agarose
• 200 ml 1x TBE buffer
• erlenmeyer flask (500 ml)
• microwave oven
• microcentrifuge tubes
• electrophoresis apparatus
• Ethidium Bromide
• gloves

Procedure

• Dissolve 2 g Agarose into 200 ml 1x TBE Buffer. This way you will obtain a 1% Agarose gel.
  
• Heat this mixture in the microwave oven for 3-4 minutes (position of the button is "cuisson"). Stir or swirl from time to time.
  
• Clamp the gel rack in the holder and add 2 drops of Ethidium Bromide. Also insert comb. Use gloves!
  
• When the melted Agarose has cooled down, you can pour it into the gel rack. Mix the EtBr with the gel using the comb. Use gloves!
  
• Wait until the Agarose is properly jellified (15 minutes).
  
• Put the gel rack with the gel inside the electrophoresis tank. Fill the tank with 1x TBE Buffer and remove the comb. (DNA moves towards the positive anode, which is the red side). Use Gloves!
  
• Now you can load the sample (25 μl). No gloves!
  
• Put the lid onto the apparatus (gloves!) and start the electrophoresis (no gloves): set-set-125V-500mA-1h-run. You should see some bubbles near the electrodes.
  
• After 1 hour, stop the electrophoresis, remove the lid and take the gel rack to the UV lamp.
  
• Take a look at the gel under UV radiation. Wear eye protection!
  
• You can cut out the part of the gel that you need for later experiments with a scalpel.
  

Restriction digest

Estimated time
unknown (2 hours + gel electrophoresis)

Materials needed

• Restriction enzymes (EcoR I, Spe I and Xba I) !! ON ICE 88
• Restiction buffer (buffer H)
• Deionized, sterile H₂O
• Plasmid DNA
• Blue Juice

Procedure
Here we describe a 20μl reaction. The used restriction enzymes are from Roche. Prepare several tubes of the same sample.

• Add the following to a microcentrifuge tube:
  • X