Protocoles

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Synthetic biology protocols

Chemocompetent bacteria

1. Inoculate 500 ml LB with 5 ml of the overnight culture and incubate it shaking at 37 °C till the OD = 0.4.
2. Cool the culture for 10 min. in some ice.
3. Centifruge the sample at 6000 rpm at 4 °C for 3 minutes.
4. Suspend the precipitate gently in ~ 20 ml of cold solution of 0.1 M CaCl2, then add 0.1 M CaCl2 to a volume of 300 ml.
5. Centrifuge again as above.
6. Suspend the precipitate in 10 ml of cold 0.1 M CaCl2 and incubate it for 30 min.
7. Centrifuge again.
8. Suspend the sample in 6 ml of 0.1 M CaCl2 and 15% glycerol and Pipette 50 μl to an eppendorf (put them immediately in liquid nitrogen) and store at -80 °C.

Transformation of chemocompetent bacteria

1. Put the bacteria into ice for 2-3 minutes (or until it melts)
2. Add a cooled plasmid or ligation in a volume not bigger than 20 μl and stir with a tip
3. Keep it in the ice for 20 to 30 minutes
4. Put it to a heating block set for 42°C for 1.5 min
5. Put it back to ice for 2 min
6. Add 900 μl of SOB and incubate at 37°C for 1 h
7. Sow on the appropriate selective deposit.

Transformation of electrocompetent bacteria

1. Put the bacteria into some ice for 2-3 minutes (or until it melts).
2. 50 μl of bacteria pipete to a dialised and cooled plasmid DNA or ligation
3. Put the transformation mixture to a cuvette (also previously cooled on ice) - it is important that the sample must be on the bottom of the cuvette and free of air bubbles. You can tap the cuvette several times on the table.
4. Transform the bacteria in an electroporator set on 2500 V (time constant should be around 5)
5. Immediately add 900 μl of SOB and incubate at 37°C for 1 h
6. Sow the appropriate selective deposit

Alkaline Lysis

1. Pick single colony and inoculate 3 ml of LB broth. Shake at 37°C overnight.
2. Centrifuge two times 1.5 ml cells in the same 1.5 ml Eppendorf tube at 6000 rpm for 1 minute. Aspirate supernatant.
3. Resuspend cell pellet in 100 μl of GTE buffer (50 mM Glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8).
4. Add 200 μl of buffer II (0.2 M NaOH, 1% SDS). Invert gently tube 6-8 times.
5. Add 150 μl of buffer III (3M potassium acetate, 2M acetic acid, pH 5.5). This solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in an insoluble, white, amorphous precipitate.
6. Incubate in ice for 5 min. Spin at top speed 10 min.
7. Transfer supernatant to a new tube, being careful not to pick up any white flakes. Precipitate the nucleic acids with 1ml of 96% ethanol for 5 minutes at room temperature and centrifuge at top speed for 10 minutes. Aspirate supernatant, add 200 &mul 70% ethanol and centrifuge at top speed for 1 minute.
8. Aspirate off all the ethanol supernatant. Dissolve the pellet in 40 μl of water. Store at -20°C.

DNA Digestion

1. Add to Eppendorf tube (for 20 μl):
• 15 μl water
• 2 μl buffer
• 2 μl DNA that you want digest (ex. plasmid)
• 0,5 μl enzyme X and 0,5 μl enzyme Y (or 1 μl enzyme Z)

(for 10 &mul [verification cloning]):
• 7 μl plasmid
• 1 μl buffer
• 1 μl water
• 0,5 μl enzyme X and 0,5 μl enzyme Y (or 1 μl enzyme Z)
2. Tubes incubate at 37°C for 1 h.

DNA Ligation

1. Add to Eppendorf tube
• 5 μl water
• 2 μl buffer
• 10 μl digest plasmids mix
• 1 μl T4 ligase
2. Incubate 2-3 h at room temperature or O/N at 16 degrees Celsius

PCR

Add to PCR tube (0,2 ml), do it on ice:

• 34 μl water
• 10 μl buffer with MgCl2
• 1 μl 10mM dNTPs
• 2 μl primers
• 2 μl template DNA (200x attenuate plasmid)
• 1 μl Phusion polymerase

PCR programme:

• Initialization step – 98°C – 3 min
• Denaturation step – 98°C – 30 sec
• Annealing step – 55°C – 30 sec
• Elongation step – 72°C – 40 sec
• Final elongation – 72°C – 5 min
• Final hold – 4°C – to end of time
• 30 cycles

Total time: about 1 h 40 minutes.