Template:NAU-CHINA/protocols
IGEM
Protocols
PCR amplification system
PCR
- Reagent
- Volume/μL
- 2× Taq master Mix
- 10
- ddh₂o
- 7
- primer F(10μM)
- 1
- primer R(10μM)
- 1
- template DNA
- 1
SOEPCR
Experimental idea
We use the primers with complementary ends to make the PCR product forms an overlapping strand, thereby splicing the amplified fragments of different sources by overlapping the overlapping strands in subsequent amplification reactions.
SOE PCR amplification system
Initial PCR: to achieve the one fragment with the terminal another fragment.
Secondary PCR: to achieve the whole fragment AB which has both fragment A and fragment B.
- Reagent
- Volume/μL
- 2× primer STAR Max
- 10
- ddh₂o
- 7
- primer a/c(10μM)
- 1
- primer X/Y(10μM)
- 1
- template DNA
- 1
- Reagent
- Volume/μL
- 2× primer STAR Max
- 10
- ddh₂o
- 7
- primer a(10μM)
- 1
- primer c(10μM)
- 1
- template DNA(A)
- 1
- template DNA(B)
- 1
Gel Extraction
1.Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE bu er or TBE bu er be used as running buffer. Do not reuse running bu er as its pH will increase and reduce yields.
2.When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose. 3.Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL.
4.Add 1 volume Binding Buffer (XP2).
5.Incubate at 50-60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
6.Insert a HiBind® DNA Mini Column in a 2 mL Collection Tube.
7.Add no more than 700 μL DNA/agarose solution from Step 5 to the HiBind® DNA Mini Column. Centrifuge at 10,000 x g for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
8.Repeat Steps 7 until all of the sample has been transferred to the column.
9.Add 300 μL Binding Buffer (XP2). Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
10.Add 700 μL SPW Wash Buffer. Centrifuge at maximum speed for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
11.Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
12.Add 50μL deionized water directly to the center of the column membrane. Centrifuge at maximum speed for 1 minute.
13.Store DNA at -20°C.
2.When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose. 3.Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL.
4.Add 1 volume Binding Buffer (XP2).
5.Incubate at 50-60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
6.Insert a HiBind® DNA Mini Column in a 2 mL Collection Tube.
7.Add no more than 700 μL DNA/agarose solution from Step 5 to the HiBind® DNA Mini Column. Centrifuge at 10,000 x g for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
8.Repeat Steps 7 until all of the sample has been transferred to the column.
9.Add 300 μL Binding Buffer (XP2). Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
10.Add 700 μL SPW Wash Buffer. Centrifuge at maximum speed for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
11.Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
12.Add 50μL deionized water directly to the center of the column membrane. Centrifuge at maximum speed for 1 minute.
13.Store DNA at -20°C.
The double enzyme digestion system
- Components (50uL)
- Volume/μL
- 10 x buffer
- 5
- Enzyme I
- 1
- EnzymeⅡ
- 1
- DNA
- 1-2μg
- ddh₂o
- Add to 50μL
DNA agarose gel electrophoresis
1. Seal the two ends of the gel-forming model with medical tape properly, position it horizontally, position the selected comb properly.
2. Weigh 0.5g agarose for DNA electrophoresis, put it in a 250 mL Erlenmeyer flask, add 50 mL 1 x TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.
3. Turn off the microwave, remove the fluid to model, let sit at room temperature until gel completely solidified. It takes about 30 minutes. Remove the tape, pull out the comb, put the plastic sheet into the electrophoresis tank.
4. Add 1 x TAE buffer into the electrophoresis tank above the gel surface around high 2mm. 5. Using pipette to transfer samples. Add mixture of 5 μL DNA sample and 2ul 10x loading buffer to spotting holes.
6. Power on, and adjust the voltage to 120 volts. After 15-20 minutes of electrophoresis, remove the gel plate, staining in ethidium bromide solution for 10min, then observe the result under UV light.
2. Weigh 0.5g agarose for DNA electrophoresis, put it in a 250 mL Erlenmeyer flask, add 50 mL 1 x TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.
3. Turn off the microwave, remove the fluid to model, let sit at room temperature until gel completely solidified. It takes about 30 minutes. Remove the tape, pull out the comb, put the plastic sheet into the electrophoresis tank.
4. Add 1 x TAE buffer into the electrophoresis tank above the gel surface around high 2mm. 5. Using pipette to transfer samples. Add mixture of 5 μL DNA sample and 2ul 10x loading buffer to spotting holes.
6. Power on, and adjust the voltage to 120 volts. After 15-20 minutes of electrophoresis, remove the gel plate, staining in ethidium bromide solution for 10min, then observe the result under UV light.
Plasmid extraction
1. Add 200 μL buffer CBS to Adsorption Column (column in the collection tube) and centrifuge at 12,000 rpm for 1 minute, discard the filtrate and reuse collection tube.
2. Take 5-10mL bacterial solution into a centrifuge tube, centrifuge at 8,000 rpm for 1 min and remove supernatant.
3. Add 250 μL SolutionⅠ in centrifuge tube, using the pipet or vortex oscillator to suspend the cells.
4. Add 250 μL Solution II in centrifuge tube and gently flip upside down for 4-6 times to make sure the germ is full cracked. 5. Add 350 μL Solution III, gently flip upside down for 8-9 times to mix until white, flocculent precipitate appears and centrifuge at 12,000rpm for 10 minutes.
6. Add the supernatant to the adsorption column in step 1, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube.
7. Add 500 μL W1 solution to the adsorption column, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube
8. Add 500 μL wash solution to the adsorption column, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube.
9. Repeat step 8.
10. Centrifuge the empty adsorption column at 12,000rpm for l minute to dry the column matrix. (Residual ethanol may impact downstream application)
11. Transfer the adsorption column into a clean 1.5 mL centrifuge tube, add 50 -100μL Elution buffer to the center of the column membrane, let sit at room temperature for 2 minutes and centrifuge at 12,000rpm for l minute, collect the plasmid solution in the centrifuge tube.
12. Store the plasmid at -20 ° C.
2. Take 5-10mL bacterial solution into a centrifuge tube, centrifuge at 8,000 rpm for 1 min and remove supernatant.
3. Add 250 μL SolutionⅠ in centrifuge tube, using the pipet or vortex oscillator to suspend the cells.
4. Add 250 μL Solution II in centrifuge tube and gently flip upside down for 4-6 times to make sure the germ is full cracked. 5. Add 350 μL Solution III, gently flip upside down for 8-9 times to mix until white, flocculent precipitate appears and centrifuge at 12,000rpm for 10 minutes.
6. Add the supernatant to the adsorption column in step 1, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube.
7. Add 500 μL W1 solution to the adsorption column, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube
8. Add 500 μL wash solution to the adsorption column, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube.
9. Repeat step 8.
10. Centrifuge the empty adsorption column at 12,000rpm for l minute to dry the column matrix. (Residual ethanol may impact downstream application)
11. Transfer the adsorption column into a clean 1.5 mL centrifuge tube, add 50 -100μL Elution buffer to the center of the column membrane, let sit at room temperature for 2 minutes and centrifuge at 12,000rpm for l minute, collect the plasmid solution in the centrifuge tube.
12. Store the plasmid at -20 ° C.
The double enzyme digestion system
- Components (10uL)
- Volume/μL
- SolutionⅠ
- 5
- Plasmid Skeleton
- 0.5
- Insert Gene
- 4.5
- Components (10uL)
- Volume/μL
- T4 DNA ligase
- 1
- 10 × T4 DNA Ligase Buffer
- 1
- Plasmid Skeleton
- 1.5
- Insert Gene
- 6.5
Transformation
prokaryotic cell(Escherichia coli)
1. Take competent cells (E.coli DH5α or BL21) from -80°C refrigerator and put it on ice. （Set negative control by using chemically competent E.coli cells without plasmids）
2. When the competent cells dissolve (about 10min), add 10 μL DNA Ligation product or 2 μL plasmid per tube, standing for 30 minutes.
3. Heat shock at 42°C for exactly 90 seconds.
4. Put the 1.5 mL tubes back on ice for 5 minutes.
5. Add 500 μL LB fluid medium without antibiotics into the 1.5 mL tubes and then culture in the shaker incubator at 37°C for 1h 20 minutes.
6. Extract 100-200 μL bacteria liquid, spread it on LB medium with relevant antibiotic.
7. Place plates upside down and incubate at 37°C overnight.
eukaryotic cell(Saccharomyces cerevisiae)
We use the kit from MP company.
1.inoculate a single colony into 100mL YPD broth and grow with creation at 30°C to mid log, OD0.6(Approximately 12-24 hours).
2.Spin to pellet cells at 400 x g for 5 minutes; discard supernatant.
3.Resuspend cells in a total of 9 mL TE, pH 7.5. Spin to pellet cells and discard supernatant.
4.Gently resuspend cells in 5 mL Lithium/Cesium Acetate Solution.
5.Incubate at 30°C for 30 minutes with gentle shaking.
6.Spin at 400 x g for 5 minutes to pellet cells and discard supernatant.
7.Gently resuspend in 1 mL TE, pH 7.5. Cells are now ready for transformation.
8.In a 1.5 mL tube combine:
Gently mix and incubate at room temperature for 15 minutes.
9.In a separate tube, combine 0.8 mL PGE and 0.2 mL TE/Cation MIXX for each transformation reaction. Add 1 mL of this PEG/TE/Cation MIXX to each transformation reaction. Mix cells into solution with gentle pipetting.
10.Incubate at 30°C for 10 minutes.
11.Heat shock at 42°C for 10 minutes; cool to 30°C.
12.Pellet cells in a micro centrifuge at 14000 x g for 5-10 seconds and remove supernatant.
13.Resuspend in 200μL SOS and plate in appropriate selective media either directly or in top agar. Incubate at 30℃ for 48-72 hours until transforming colonies appear.
1. Take competent cells (E.coli DH5α or BL21) from -80°C refrigerator and put it on ice. （Set negative control by using chemically competent E.coli cells without plasmids）
2. When the competent cells dissolve (about 10min), add 10 μL DNA Ligation product or 2 μL plasmid per tube, standing for 30 minutes.
3. Heat shock at 42°C for exactly 90 seconds.
4. Put the 1.5 mL tubes back on ice for 5 minutes.
5. Add 500 μL LB fluid medium without antibiotics into the 1.5 mL tubes and then culture in the shaker incubator at 37°C for 1h 20 minutes.
6. Extract 100-200 μL bacteria liquid, spread it on LB medium with relevant antibiotic.
7. Place plates upside down and incubate at 37°C overnight.
eukaryotic cell(Saccharomyces cerevisiae)
We use the kit from MP company.
1.inoculate a single colony into 100mL YPD broth and grow with creation at 30°C to mid log, OD0.6(Approximately 12-24 hours).
2.Spin to pellet cells at 400 x g for 5 minutes; discard supernatant.
3.Resuspend cells in a total of 9 mL TE, pH 7.5. Spin to pellet cells and discard supernatant.
4.Gently resuspend cells in 5 mL Lithium/Cesium Acetate Solution.
5.Incubate at 30°C for 30 minutes with gentle shaking.
6.Spin at 400 x g for 5 minutes to pellet cells and discard supernatant.
7.Gently resuspend in 1 mL TE, pH 7.5. Cells are now ready for transformation.
8.In a 1.5 mL tube combine:
- Components (10uL)
- Volume/μL
- yeast cells
- 100
- Carrier DNA
- 5
- Histamine Solution
- 5
- plasmid DNA
- less than 10(total 0.01-1.0μg)
Gently mix and incubate at room temperature for 15 minutes.
9.In a separate tube, combine 0.8 mL PGE and 0.2 mL TE/Cation MIXX for each transformation reaction. Add 1 mL of this PEG/TE/Cation MIXX to each transformation reaction. Mix cells into solution with gentle pipetting.
10.Incubate at 30°C for 10 minutes.
11.Heat shock at 42°C for 10 minutes; cool to 30°C.
12.Pellet cells in a micro centrifuge at 14000 x g for 5-10 seconds and remove supernatant.
13.Resuspend in 200μL SOS and plate in appropriate selective media either directly or in top agar. Incubate at 30℃ for 48-72 hours until transforming colonies appear.
Yeast genome extractionn
1.Pour 2mL culture into 2 mL EP tubes and pellet cells in centrifuge at 12000 rpm for 2 minutes and remove supernatant.
2.Add 500 μL CTAB into the EP tubes and use boiling water boil the mixture more than 10 minutes.
3.Add 500 μL phenolic isopentanol and shake the tube fiercely.
4.Pellet the mixture in centrifuge at 12000 rpm for 2 minutes and remove supernatant into a new 2mL EP tube.
5.Repeat Step 3 to 4 but remove supernatant into a new 1.5mL EP tube.
6.Add 2/3 volume Isopropyl alcohol and shake the tube fiercely then store at -20℃ more than 2 hours.
7.Centrifuge at 12000 rpm for 2 minutes discard the supernatant.
8.Add 70% ethanol 500μL into the tube and centrifuge at 12000 rpm for 2 minutes discard the supernatant.
9.Use the hair dryer to dry the sample(Note: do not dry all the liquid in tube).
10.Add 30-70μL ddh₂o into the tube to dissolve the genome.
11.Store the sample at -20 °C.
2.Add 500 μL CTAB into the EP tubes and use boiling water boil the mixture more than 10 minutes.
3.Add 500 μL phenolic isopentanol and shake the tube fiercely.
4.Pellet the mixture in centrifuge at 12000 rpm for 2 minutes and remove supernatant into a new 2mL EP tube.
5.Repeat Step 3 to 4 but remove supernatant into a new 1.5mL EP tube.
6.Add 2/3 volume Isopropyl alcohol and shake the tube fiercely then store at -20℃ more than 2 hours.
7.Centrifuge at 12000 rpm for 2 minutes discard the supernatant.
8.Add 70% ethanol 500μL into the tube and centrifuge at 12000 rpm for 2 minutes discard the supernatant.
9.Use the hair dryer to dry the sample(Note: do not dry all the liquid in tube).
10.Add 30-70μL ddh₂o into the tube to dissolve the genome.
11.Store the sample at -20 °C.
High performance liquid
chromatography
chromatography
Instrument:
DIONEX UltiMate 3000 UHPLC , Buchner funnel , Vacuum suction machine,Organic membrane , 1000ml cylinder , 100ml cylinder , ultrasonic oscillator Reagent:
Methanol(chromatographic pure) acetonitrile(chromatographic pure) acetic acid(chromatographic pure) Ultra pure water
Steps for preparing mobile phase:
Step one: Pour 1000ml of acetonitrile into a 1000ml screw mouth
bottle. Pouring 990ml of water and 10ml of acetic acid into a 1000ml
screw mouth bottle.
Step two: Oscillating bottle up and down.
Step three: Use 50ml of mobile phase to rinse the buchner funnel.
(Organic waste liquid is poured into the waste liquid recovery barrel)
Step four: Use buchner funnel and organic membrane to filter the
mobile phase.
Step five: Use ultrasonic oscillator to oscillat the bottle to remove
DIONEX UltiMate 3000 UHPLC , Buchner funnel , Vacuum suction machine,Organic membrane , 1000ml cylinder , 100ml cylinder , ultrasonic oscillator Reagent:
Methanol(chromatographic pure) acetonitrile(chromatographic pure) acetic acid(chromatographic pure) Ultra pure water
Steps for preparing mobile phase:
Step one: Pour 1000ml of acetonitrile into a 1000ml screw mouth
bottle. Pouring 990ml of water and 10ml of acetic acid into a 1000ml
screw mouth bottle.
Step two: Oscillating bottle up and down.
Step three: Use 50ml of mobile phase to rinse the buchner funnel.
(Organic waste liquid is poured into the waste liquid recovery barrel)
Step four: Use buchner funnel and organic membrane to filter the
mobile phase.
Step five: Use ultrasonic oscillator to oscillat the bottle to remove
the gas in the mobile phase.
Step six: Store the mobile phase at room temperature for using.
Steps for extracting sample:
Step one: Pipet 2ml of liquid medium into a 10ml centrifuge tube.
Step two: Add 2ml of Methanol into the centrifuge tube.
Step three: Use vortex to blend the mixture.
Step four: Centrifuge at 12000×rpm for 5 minutes.
Step five: Pipet 2ml of liquid supernatant into a 2ml of centrifuge
tube.
Step six: Use organic membrane to filter the sample.
Step seven: Repeat the step six.
Step eight: Store the sample at room temperature.
Steps for using HPLC (High performance liquid chromatography):
Step one: Open the instrument before opening the computer.
Step two: Loosen instrument valve.
Step three: Use 50% of acetonitrile rinsing the chromatographic column for about 30 minutes until there is no impurity to interfere baseline.
Step four: Close the valve.
Step five: Monitore baseline,.
Step six: Create method file and sequence file.
Step seven: Start sampling when the baseline is flat.(When testing
3-pba, we use light of 240nm and 280nm. The current speed is 0.8
ml/min.)
Step eight: Rinse the column with 100% acetonitrile for at least 40
minutes.
Step nine: Turn off the device in reverse order when the samples have
all be detected.
Step ten: Cleaning waste liquid.
