Team:KULeuven/Protocols

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Protocols

Transformation of E. Coli

1. Preparing a cell culture
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 μg/ml) streptomycine (20 μg/ml) LB-agar plate and grow overnight
• pick a single colony and grow overnight in 3 ml LB at 37 °C
• inoculate 100 ml LB with 1 ml of the overnight culture and grow at 37 °C for 2-3 hours (OD₆₀₀=0.5)

2. CaCL₂ method

• collect the bacterial cells by centrifugation (6000 rpm, 5 min, 4 °C)
• 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, 4 °C
• resuspend the pellet in 1 ml 0.1 M CaCL₂
• use immediately or store at -70 °C with addition of glycerol to a final concentration of 15%
• add 100 μl competent cells to 20 μl ice cold ligation mix in a microcentrifuge tube
• keep on ice for at least 15 min.
• heat shock for 25 sec. at 42 °C and place on ice for 2 min.
• add 1 ml LB and incubate while shaking at 37 °C for 40 min.
• plate 100 μl and 900 μl (spin down and resuspend in 100 μl) on seperate LB plates with the required antibiotic (pGEM, pUC, all yeast shuttle vectors: 100 μg/ml ampicilline)
• incubate overnight at 37 °C

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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.

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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 μl plasmid DNA (approximately 500 ng -calculate volume!!).
  • (16-X) μl deionized, sterile H₂O.
  • 2 μl Buffer H. Vortex buffer before pipetting to ensure that it is well-mixed.
  • 1 μl EcoR I and 1 μl Spe I or Xba I. The enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μl, just touch your tip to the surface of the liquid when pipetting. The restriction enzymes must be the last thing you add. Always keep them on ice.
    
• Incubate the tubes at 37 °C for 1,5 - 2 hours (heat block or oven). In the meantime, you can prepare the Agaros gel.
  
• Add 4 μl Blue juice to each tube.
  
• Load 5 μl reference mixture (ladder) onto the gel.
  
• Load 22 μl digest onto the gel. Make sure that you have multiple lanes with the same BioBrick (higher concentration).
  
• Start the electrophoresis (this should take about 1 hour). Look at the result under UV-light and cut out the correct fragment.
  
• Purify the obtained fragment (with other kit - see manual).

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Plasmid DNA preparation

(Minipreps 1 - 2 ml of culture)

CTAB Method

Del Sal, G. et al. (1988) Nucleic Acids Research 16(20):9878
This method gives enough DNA to perform 10 - 50 regular digests (depending on the copy number of the plasmid) and is qualitatively pure enough for dsDNA sequencing. This method can readily be scaled up to 100 ml cultures.

Buffers:

• STET: 8% w/v sucrose, 0,1% v/v Triton X-100 (add after solution has been autoclaved), 50mM EDTA, 50mM Tris-HcL pH 8,0
• CTAB: 5% w/v cetyl trimethyl ammonium bromice (precipitates below 20 °C, reheat to dissolve)
• Lysozyme: 50 ^mg/_ml (can be stored in water at -20 °C)
• NaCl: 1,2M
• TE: 1mM EDTA, 10mM Tris-HCl pH 8,0
• EtOH: 100% and 70% ethanol, reheat to dissolve)
• Lysozyme: 50 ^mg/_ml (can be stored in water at -20 °C)
• NaCl: 1,2M
• TE: 1mM EDTA, 10mM Tris-HCl pH 8,0
• EtOH: 100% and 70% ethanol

Method:

• Preculture: 4 ml LB + antibiotic (overnight at 37 °C)
• Pellet 1,5 ml bacterial cells (centrifuge 5 min on 13000 rpm
• Resuspend cells in 300 μl STET plus 3 μl of 50 ^mg/_ml lysozyme (make mix for all preps beforehand)
• Incubate 5 min at RT
• Heat to 95 °C or 100 °C for 1 minute
• Spin (13000 rpm, 10 - 15 min)
• Remove pellet (toothpick or 200 μl pipet
• Add 6 μl CTAB, vortex briefly
• Spin (13000 rpm), remove liquid
• Resuspend pellet in 300 μl 1,2 M NaCl )requires vigorous vortexing and/or pipetting with pipet tip)
• Add 750 μl EtOH 100%
• Spin (13000 rpm, 5 - 10 min), discard liquid
• EtOH 70%
• Centrifuge 5 min, 13000 rpm, discard liquid
• Spin again briefly to collect all liquid at the bottom, remove all liquid with pipet
• Leave tubes open for about 5 min to air-dry pellet
• resuspend in 50 or 30 μl TE, use 1 - 2 μl for digestion with restriction enzyme(s)

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