"
Team:Hong Kong CUHK/protocol
From 2013.igem.org
Project Overview
1. Cloning
1.1 Gene Amplification with PCR
1.2 PCR Purification ( Using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0)
1.3 Overlapping PCR
1.4 Agarose Gel Electrophoresis
1.5 Preparation of Competent Cells
1.6 Bacterial Transformation
1.7 Colony PCR
1.8 Inoculation
1.9 Plasmid DNA Extraction (Using TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0)
1.10 Double Digestion
1.11 Gel Extraction
1.12 Ligation
2. Functional test
2.1 Cell Viability Test with Voltage Applied
2.2 Cell Viability Test with BaP
1.1 Gene Amplification with PCR
2. Add the following components into each reaction tube:
- 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 5–10 seconds
72°C 15–30 seconds/kb
Final extension:
72°C 5–10 minutes
Hold:
4°C
1.2 PCR Purification ( Using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0)
2. Add 3X sample volume’s Buffer GM to the eppendorf containing PCR product. Mix it well and transfer the mixture into the Spin Column and centrifuge at 12,000 rpm for 1 min. For improvement the recovery rate of DNA, transfer the flow-through to Spin Column again and centrifuge again. Discard the flow-through.
3. 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.
4. Repeat Step 10.
5. Place the Spin Column back into Collection Tube. Centrifuge at 12,000 rpm for 1 minute.
6. 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.
7. Take back the flow-through to the center of the membrane. Let it stand for 1 minute at room temperature and then centrifuge again.
1.3 Overlapping PCR
2. Add the following components into each reaction tube:
- 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
1.4 Agarose Gel Electrophoresis
1. Dissolve 0.55 g agarose into 55 ml 0.5X TBE buffer.
2. Microwave (high power, 800W) for 1 min.
3. Cool it down using running water for 1 min.
4. Add 1 μl GelRed
5. Pour the solution to tightened tank with gates and gel comb and allow it to solidify.
6. Transfer the gel to gel tank once it becomes solid.
B. Run gel
1. Orient the gel with wells facing the black negative electrode. Check if the gel is covered by TBE buffer in the tank. If not, add TBE buffer to cover it to about 1mm.
2. Mix loading dye and the insert/plasmid before adding to the wells. For example, if the DNA we have got is 45μl, and the loading dye we have got is 10X, then add 5μl of loading dye to the samples. Mixture should be in blue.
3. To run the gel, add all samples to the wells of gel. Then add 1kb DNA ladder to a separate well. 1μl should be enough for detection under UV.
4. Connect the electrodes to the power supply with correct colour. Set the power supply to 120V. Check if there are bubbles on the negative electrodes.
5. Allow it to run for about 15-30min. To avoid running the band off the gel, the yellow band (position of the smallest fragments) should stay on the gel.
1.5 Preparation of Competent Cells
1. Autoclave glassware filled with 3/4 dd-H2O to remove most residual detergent.
2. Autoclave media and buffers in detergent-free glassware
B. Preparation of the competent cells
Reagents:
- Glycerol stock
- LB plate
- MgCl2-CaCl2 solution
- MgCl2‧6H2O 3.25g
- CaCl2‧2H2O 0.6g
- Add H2O to 200 ml
- 100mM CaCl2
- CaCl2‧2H2O 2.95g
- Add H2O to 200 ml
- 80% glycerol
- Liquid nitrogen
Procedure:
Day 1
1. Flame the metal inoculating loop until it is red got and then cools it down.
2. Scrape off a portion from the top of the frozen glycerol stock [DO NOT THAW].
3. Streak it onto the LB plate.
4. Put the stock back to -80 oC immediately.
5. Leave the plates for 5 min and place them upside down in the 37oC incubator for 16-20 h.
Day 2
6. Pick a single colony into 5 ml of LB medium.
7. Inoculate the culture overnight at 37oC with shaking at 250 rpm.
Day 3
8. Inoculate 100 ml LB medium with 1 ml of saturated overnight culture.
9. Shake at 37oC until OD600 = 0.4 (usually 2-3 h).
10. Place in an ice bath for 10 min.
[After this point, the cells must be placed on ice!]
11. Pre-cool solution, centrifuge, pipette tips, falcon, and eppendorf.
12. Transfer the culture into two pre-chilled 50ml falcon.
13. Centrifuge at 2700 x g for 10 min at 4oC
14. Remove the medium, and resuspend the cell pellet with 1.6 ml ice-cold 100 mM CaCl2 by swirling on ice gently.
15. Incubate on ice for 30 min.
16. Centrifuge at 2700 x g for 10 minutes at 4oC.
17. Remove the medium, and resuspend the cell pellet with 1.6 ml ice-cold 100 mM CaCl2 by swirling on ice gently.
18. Incubate on ice for 20 min.
19. Pool all cells into one tube and add 0.5 ml ice-cold 80% glycerol and swirl to mix.
20. Freeze 100 μl aliquots in liquid nitrogen.
21. Store in -80oC.
1.6 Bacterial Transformation
2. Add 50 - 100 ng DNA to competent cell culture.
3. Put in ice for 10min
4. Heat shock at 42oC for 1.5-2 min
5. Put in ice for 2 min.
6. Add 1 ml LB medium.
7. Incubate at 37oC for 30-90 min with shaking (~ 250 rpm).
8. Spread plate (with suitable antibiotics)
9. Spin down the remaining cells and discard large amount supernatant (1 ml).
10. Resuspend the cell pellet and spread plate.
11. Incubate at 37oC overnight (preferably ~16 – 24h).
1.7Colony PCR
2. Add the following components into each PCR tube:
Acutoclaved water 2.3μl
rTaq 2.5μl
Primers(BBa-G1004F+BBa-G1005R) 0.1μl +0.1μl
3. Pick Colonies into each PCR tube. Be careful to label it well.
3. Mix well and run the following program:
Heatlid
104°C
Initial denaturation:
94°C 5min
20–25 cycles
94°C 30 seconds
63°C 30 seconds
Final extension:
72°C 1min10sec
Hold:
4°C
1.8 Inoculation
2. Incubate at 37oC for 12 - 16 h with shaking at 250 rpm.
1.9 Plasmid DNA Preparation (using TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0)
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.
1.10 Restriction Digestion
15μl Insert/Vector# + ddH2O*
2 μl 10X NEB Buffer2
2 μl 10X BSA
0.5 μl Enzyme 1
0.5 μl Enzyme 2
# At least 200ng DNA should be added
* Water is added first and the template the last
2. Incubate the reaction mixture at 37oC for 1 h.
Buffer Chart
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp
1.11 Gel Extraction ( Using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0)
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.
1.12 Ligation
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.
2.1 Cell Viability Test with Voltage applied
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’
2.2 Cell Viability Test with BaP
- 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. - 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. - Shake the cells in 25oC for 12 hours.
- Take another reading of OD600.
- Repeat Step 6-7.
- Plot a growth curve of cells under different concentrations of BaP/Quinone
