Team:KULeuven/Protocols

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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optimizing DNA purification & ligation

Gel (STOCK)
1 % gel with low melting agarose dissolved in 1X TAE. Stored in the fridge.

Making the construct
The comb is put on the plate (1) and held in place on with clips on either side of the comb (2). After adding the clips it is important to check that the comb is still touching the top of the plate. Weights are also added, in the form of falcons filled with a liquid, to keep the construct in place (3).





Adding the gel to the construct
Before use, heat up the gel till it simmers. Afterwards the gel is left for 30 min to cool down. After 30 minutes the gel should have cooled down enough to make the pipetting onto the construct easier. Take care to let the solution flow at a constant speed out of the pipette tip. For an optimal result, 6-9mL of solution should be added to the construct and then be left to solidify for roughly 30 minutes (4).

Dismantling the construct
The weights are lifted from the clips. Next, one of the clips is removed, during the removal it is necessary to hold the comb in place by pushing down on it (5). The comb is then removed with the clip still attached to it. It is gently pulled out of the gel with one hand whilst the other hand keeps the plate on the bench by pushing down on the plate (6). After removing the comb, the samples (digested DNA) can be loaded on to the gel (7).

Ligation
After the gel run, the desired bands are excised and used in the ligation protocol. Ligation was done using the rapid ligation kit from Roche (Cat. No. 11 635 379 001). First you need to add 2µL nuclease free H2O into the Eppendorf tube. This is followed by 2µL of DNA Dilution Buffer from the kit. The next steps need to be executed rapidly. Put the vector and its insert in a heat block on 70°C for 2 minutes. For the control, use nuclease free H2O instead of an insert. Pipet 0.5µL vector and 5.5µL insert into the Eppendorf tube. This must immediately be followed by 10µL of T4 DNA Ligation Buffer and 1µL of T4 DNA Ligase, 5 U/μl. Keep the samples at room temperature for 10 minutes, then add 50µL competent cells to each eppendorf tube. Keep the samples on ice for 30 minutes, after which the cells are heat shocked at 42°C for 45s. Finally, put the samples at room temperature, add 100µL of LB medium and plate the sample on an agar plate with ampicillin. Towards the end of our project, we made an additional optimization. After excising the desired band from the gel, we dissolved the low-melting agarose, using the enzyme agarase according to the manufacturer’s instructions (New England Biolabs, Cat. No. M0392). As the solution will not solidify anymore after this treatment, ligations can be done for a more extended period. We used the agarase-treated solutions to perform ligations at 16°C for 1 hour with standard T4 DNA ligase (Roche Cat. No. 10716359001), which resulted in very nice yields!


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