Team:Hong Kong CUHK/protocol

• 5x Phusion HF Buffer* (1X final concentration is recommended)
• 10 mM dNTPs (200 μM final concentration is recommended)
• Primer solution (0.5 μM final concentration of each is recommended)
• DNA Template
• Phusion DNA Polymerase (0.02 U/μl final concentration is recommended)

3. Mix well and run the following program:
2-step PCR Cycling Program
Initial denaturation:
98°C   30 seconds
25–35 cycles:
98°C   10 seconds (denaturation)
60°C  10 seconds (annealing)
72°C   15–30 seconds/kb (extension)
Hold:
4°C
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1.4 Agarose Gel Electrophoresis

1.5 Preparation of Competent Cells

1.6 Bacterial Transformation

1.7Colony PCR

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1.8 Inoculation

1. Pick colonies and culture in 3-5 ml LB broth with antibiotics. (1μl/1ml)
2. Incubate at 37oC for 12 - 16 h with shaking at 250 rpm.

1.         Use 1 - 4 ml oft heE. coli culture. Centrifuge at 12,000 rpm for 2 minutes to harvest
the cell. Discard the supernatant.
2.         Add 250μl Solution l (containing RNaseA). Resuspend the bacterial cell pellet completely by vortexing or pipetting up and down.
Note:    Be sure thatthe bacteria are completely resuspended by vortexing and no
cell clumps remain before addition of Solution ll.
3.          Add 250μl Solution ll, and mix gently by inverting the tube 5 - 6 times to completely lysis the cell untilthe solution becomes viscous and slightly clear.
Note:    Do not allowthe lysis reaction to proceed more than 5 minutes.
4.         Add 350μl of 4℃precooling Solution lll, and mix immediately and thoroughly by
inverting the tube 5 - 6 times until a compactwhite pellet has been formed. Incubate atroom temperature for 2 minutes.
5.         Centrifuge at 12,000 rpm atroom temperature for 10 minutes.
Note:    Centrifuging at 4℃is notrecommended for precipitation.
6.         Place a Spin Column in a Collection Tube.
7.         Apply the supernatantfrom Step 6 onto the Spin Column by decant or pipetting.
Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.
8.         Pipette 500μl of Buffer WAonto the Spin Column. Centrifuge at 12,000 rpm for 30
seconds. Discard the flow-through.
9.         Pipette 700μl of Buffer WB onto the Spin Column. Centrifuge at 12,000 rpm for 30
seconds. Discard the flow-through.
Note:    Make sure thatthe amount of 100% ethanol indicated on the bottle label has
been added to Buffer WB.
10.        Repeat Step 10.
11.        Place the Spin Column back into the Collection Tube. Centrifuge at 12,000 rpm for
an additional 1 minute to remove residual wash Buffer WB.
Note:    Residual ethanol from Buffer WB may inhibit subsequent enzymatic reaction.
12.        Place the Spin Column in a new clean 1.5 ml tube.
Add 25μl Elution Buffer or sterile 60℃distilled water to the center of the Spin Column
membrane. Incubate for 1 minute atroom temperature. Centrifuge at 12,000 rpm for 1 minute to elute DNA
13.        Repeat step 12.

1.Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Cut the gel into small pieces by cutting the gel
3. Weigh the gel pieces and calculate the volume of gel. 1 mg of gel is equivalent to a
1 μl volume.
4. Add Buffer GM to gel for melting. The amount of Buffer GM is shown in the table
below.
gel concentration Buffer GM
1.0％ 3X sample volume
1.0 - 1.5％ 4X sample volume
1.5 - 2.0％ 5X sample volume
5. Mix well and melt the gel at room temperature (15 - 25℃). If the concentration of
gel is too high or the gel is hard to melt, warm at 37℃. Intermittent vortexing will
accelerate gel solubilization.
Note: Gel must be completely dissolved, or the DNA fragment recovery will be
reduced. Extend the melting time when the gel concentration is high.
6. Set a Spin Column into Collection Tube.
7. Transfer the solubilized agarose from Step 7 into the column. Centrifuge at 12,000 rpm
for 1 minute. Discard the flow-through.For improvement the recovery rate of DNA, transfer the flow-through to SpinColumn again and centrifuge again.
8. Add 700 μl of Buffer WB into the Spin Column. Centrifuge at 12,000 rpm for 30 seconds.
Discard the flow-through.
Note: Make sure that the amount of 100% ethanol specified on the bottle label has
been added to the Buffer WB.
9. Repeat Step 10.
10. Place the Spin Column back into Collection Tube. Centrifuge at 12,000 rpm for 1 minute.
11. Place the Spin Column into a new 1.5 ml tube. Add 20 μl of 60℃ sterile
distilled water to the center of the membrane. Let it stand for 1 minute at room temperature and then centrifuge.
12.Take back the flow-through to the center of the membrane. Let it stand for 1 minute at room temperature and then centrifuge again.

1. Mix the components as follows to prepare a 10 μl reaction mixture:
   0.5 μl         Water
   1 μl        10X ligation buffer
   0.5 μl        T4 DNA ligase
   2 μl        Vector
   6 μl        Insert
2. Incubate the reactions at 16oC overnight, 22oC for 1 h or stand in RT for 10min.

We are applying continuous DC Voltage to the samples by using batteries.

• Cells used: BL21 transformed with 2µL PSB1C3 + RFP

        Spread on Chloramphenicol agar plate and incubate overnight in 37 °C

• Pick 8 clones into tubes with 10 mL of LB solution.  Add 10 µL of Chloramphenicol to experimental group tubes. Do overnight shake in incubator for 16 hours to make the cells saturated.

• After 16 hours, take out the tubes from the incubator.

• Normalize the samples to around 400 – 600 ABS with OD check (using 600nm wavelength) by adding extra LB + Chloramphenicol.

• Place 5 mL of sample to each experimental tube. (6 voltages x 3 = 18 tubes)

• Next, we perform the experiment with voltage applied to each tubes.  We use one 9V battery for 9V, two 9V batteries in series for 18 V, three 9V batteries in series for 27 V, four 9V batteries in series for 36 V, and five 9V batteries in series for 45 V. Tubes will be shaken under 250 rpm and 25°C for 24 hours. For each voltage, we have three samples.

Fig. 1 +9V circuit                   Fig. 2 +18V circuit

Fig. 3 +27V circuit                         Fig. 4 +36V circuit

Fig 5. +45V circuit

• After 24 hours, we take the tubes out. Then we take 5 µL of each sample to perform serial dilution. We performed 10X, 100X, 1000X and 10,000X dilution.

• Take 1 µL from each diluted solution to the Chloramphenicol agar ‘plate table’ and streak on the square carefully. Then incubate the plates for 12 hours in 37 °C.

The ‘Plate Table’

1.  Transform BL21 cells with pSB1C3-RFP
   2.  Pick a clone for overnight culture at 37oC
   3.  Label in triplicate 12 5ml centrifuge tubes, 6 tubes of different concentrations of BaP, the other 6 tubes of different concentrations of quinone.
2.  To each of the tubes, add in 750ul of LB solution, 150ul of cells, and 100ul of BaP/Quinone solutions in 10X stock corresponding to the labels
   5.  Mix well and take an OD600 reading using a microplate reader.
3.  Shake the cells in 25oC for 12 hours.
4.  Take another reading of OD600.
5.  Repeat Step 6-7.
6.  Plot a growth curve of cells under different concentrations of BaP/Quinone