Restriction Endonuclease Reaction
1. Solutions are mixed in 50μl two pipettes and centrifugalized.
2. Pipettes are warmed in the PCR for 1 hour at 37 degrees for restriction to take place and the enzyme are
denatured 65 degrees for 20 minutes.
Team:ECNUAS/Experiments
Experiments
Enzyme digestion
Electrophoresis
DNA Agarose Gel Electrophoresis Identification
1. Configurate 1X TAE buffer.
2. Configurate and make electrophoresis gel.
3. Configurate DNA sample and marker.
4. Put the gel in appropriate place in the tank with 1X TAE buffer and sample the DNA sample and marker onto the electrophoresis gel.
5. Run the electrophoresis.
Observe the DNA stripe using Ultra-thin ultraviolet platform.
2. Configurate and make electrophoresis gel.
3. Configurate DNA sample and marker.
4. Put the gel in appropriate place in the tank with 1X TAE buffer and sample the DNA sample and marker onto the electrophoresis gel.
5. Run the electrophoresis.
Observe the DNA stripe using Ultra-thin ultraviolet platform.
DNA Agarose Gel Recovery and Purification
1. Cut down the parts of gel which contains the correct DNA and calculate the weight difference in the EP
tube from adding the gel by weighing the tube before and after adding the gel.
2. Add buffer DE-A, 3X gel volume, heat in water bath at 75 degrees until the gel melts.
3. Add buffer DE-B, 1.5X gel volume, and mix the solution until it is a transparent yellow color.
4. Transfer the liquid into a DNA preparation tube, centrifugalize at 12000 xg for 1 minute, and remove the liquid below.
5. Add 500μl Buffer W1, centrifugalize at 12000 xg for 30 seconds, and remove the liquid below.
6. Add 700μl Buffer W1, centrifugalize at 12000 xg for 30 seconds, and remove the liquid below. Repeat this process.
7. Centrifugalize at 12000 xg for 1 minute.
8. Add 25μl deionized water, which is pre-heated to 65 degrees, keep the tube in room temperature for 1 minute, and centrifugalize at 12000 xg for 1 minute.
2. Add buffer DE-A, 3X gel volume, heat in water bath at 75 degrees until the gel melts.
3. Add buffer DE-B, 1.5X gel volume, and mix the solution until it is a transparent yellow color.
4. Transfer the liquid into a DNA preparation tube, centrifugalize at 12000 xg for 1 minute, and remove the liquid below.
5. Add 500μl Buffer W1, centrifugalize at 12000 xg for 30 seconds, and remove the liquid below.
6. Add 700μl Buffer W1, centrifugalize at 12000 xg for 30 seconds, and remove the liquid below. Repeat this process.
7. Centrifugalize at 12000 xg for 1 minute.
8. Add 25μl deionized water, which is pre-heated to 65 degrees, keep the tube in room temperature for 1 minute, and centrifugalize at 12000 xg for 1 minute.
T4 ligation
DNA Clip Connection Reaction
1. Configurate DNA clip connection sample.
2. Centrifugalize and react in room temperature for 10 minutes.
3. Denature the enzyme at 65 degrees for 10 minutes.
1. Configurate DNA clip connection sample.
2. Centrifugalize and react in room temperature for 10 minutes.
3. Denature the enzyme at 65 degrees for 10 minutes.
Prepare reagents
Plasmid Transformation
1. Configurate LB solid medium, sterilize, cool, add ampicillin and kanamycin to each portion, spread it in
the plates, and wait for solidification.
2. Mix 2μL plasmid DNA and 20μl competent E. coli cell and put the tube on ice for 30 minutes.
3. Water bath the tube at 42 degrees for 90 seconds and ice bath for 2.5 minutes.
4. Add 1 mL resistance-free LB liquid medium to the tube and put it in the incubator at 37 degrees with 200 rpm for 45 minutes.
5. Centrifugalize for 2 minutes at 5000 rpm Remove 800μL supernatant. Resuspend the rest. Inoculate on the plate, and incubate at 37 degrees for one night.
2. Mix 2μL plasmid DNA and 20μl competent E. coli cell and put the tube on ice for 30 minutes.
3. Water bath the tube at 42 degrees for 90 seconds and ice bath for 2.5 minutes.
4. Add 1 mL resistance-free LB liquid medium to the tube and put it in the incubator at 37 degrees with 200 rpm for 45 minutes.
5. Centrifugalize for 2 minutes at 5000 rpm Remove 800μL supernatant. Resuspend the rest. Inoculate on the plate, and incubate at 37 degrees for one night.
Plasmid Extraction
1. Pick up monoclonal colonies on the plate, inoculate in 10 mL LB liquid medium, and cultivate in incubator
for 12 hours to 16 hours at 250 rpm.
2. Add 500μL P1 to the CP3 adsorption column, centrifugalize at 12000 rpm for 1 minute to activate the silicon membrane.
3. Take 1mL bacterial liquid. Centrifugate for 1 minute at 12000 rpm, and remove the supernatant.
4. Add 250μL P1 and resuspend the precipitate.
5. Add 250μL P2 and gently overturn the tube for 6 to 8 times.
6. Add 350μL P3. Gently overturn the tube for 6 to 8 times, and centrifugate for 10 minutes at 12000 rpm after the white precipitates form.
7. Collect the supernatant to the column. Centrifugate for 30 to 60 seconds at 12000 rpm, and remove the liquid.
8. Add 600μL PW. Centrifugate for 30 to 60 seconds at 12000 rpm. Remove the liquid, and repeat the process.
9. Centrifugate at 12000 rpm for 2 minutes.
10. Put the column into a new centrifuge tube, add 100μL EB. Keep the tube in room temperature for 2 minutes, and centrifugate at 12000 rpm for 2 minutes.
2. Add 500μL P1 to the CP3 adsorption column, centrifugalize at 12000 rpm for 1 minute to activate the silicon membrane.
3. Take 1mL bacterial liquid. Centrifugate for 1 minute at 12000 rpm, and remove the supernatant.
4. Add 250μL P1 and resuspend the precipitate.
5. Add 250μL P2 and gently overturn the tube for 6 to 8 times.
6. Add 350μL P3. Gently overturn the tube for 6 to 8 times, and centrifugate for 10 minutes at 12000 rpm after the white precipitates form.
7. Collect the supernatant to the column. Centrifugate for 30 to 60 seconds at 12000 rpm, and remove the liquid.
8. Add 600μL PW. Centrifugate for 30 to 60 seconds at 12000 rpm. Remove the liquid, and repeat the process.
9. Centrifugate at 12000 rpm for 2 minutes.
10. Put the column into a new centrifuge tube, add 100μL EB. Keep the tube in room temperature for 2 minutes, and centrifugate at 12000 rpm for 2 minutes.
Colony PCR
1. Initial denaturation takes place in PCR for 5 minutes. Denaturation, annealing, and extension repeat 34
times. The final extension and the preservation ends the PCR process in 5 minutes.
2. Run the electrophoresis to test for the positive result.
3. Select the positive colonies and enlarge cultivation and add 5 milliliters medium to each tube.
4. Extract the plasmid and send them to the sequencing company.
2. Run the electrophoresis to test for the positive result.
3. Select the positive colonies and enlarge cultivation and add 5 milliliters medium to each tube.
4. Extract the plasmid and send them to the sequencing company.
Double-Antibiotics Medium Screening
1. Prepare fresh LB medium with two antibotics (kana&) and the concentrations are kan 50 ug/ml; amp
100ug/ml.
2. Extract plasmid A and plasmid B and transform into DH5a (Ref. Plasmid Extraction & Transformation ) and culture in the double-antibiotics LB medium we prepared.
2. Extract plasmid A and plasmid B and transform into DH5a (Ref. Plasmid Extraction & Transformation ) and culture in the double-antibiotics LB medium we prepared.
Function Test
1. Prepare the cyanuric acid solution
Take 103mg cyanuric acid powder, add 8ml ddH2O, 80°C water-bath to complete dissolution, and then obtain 100mm/L solution.
2. Functional test of bacteria C
Bacteria C was cultured and induced in a certain concentration of CYA and the fluorescence intensity would be measured and collected.
The blank control in this experiment is the pure DH5a without any plasmids transformed.
3. Steps:
1）Select monoclonal C into 3ml LB medium (two antibiotics have been added) and is cultured in a constant temperature shaker at 30 °C for 8-12 hours.
2）Dilute the seed by 100 times to 50 ml of new LB medium at 30 °C until the initial OD600 reaches 0.2
3）By adding CYA to different sample tubes to a certain concentration, observing at a constant temperature 30°C for a certain period of time, the fluorescence intensity was measured by Thermo instrument (excitation wavelength is 503nm and the emission wavelength is 512nm), so as to explore the best induction condition and the response time range of the biosensor
4）Notes: during the pre-experiments, we notice that the LB medium had a serious background effect in measuring the fluorescence intensity, so all samples were measured after washing and resuspension.
Take 103mg cyanuric acid powder, add 8ml ddH2O, 80°C water-bath to complete dissolution, and then obtain 100mm/L solution.
2. Functional test of bacteria C
Bacteria C was cultured and induced in a certain concentration of CYA and the fluorescence intensity would be measured and collected.
The blank control in this experiment is the pure DH5a without any plasmids transformed.
3. Steps:
1）Select monoclonal C into 3ml LB medium (two antibiotics have been added) and is cultured in a constant temperature shaker at 30 °C for 8-12 hours.
2）Dilute the seed by 100 times to 50 ml of new LB medium at 30 °C until the initial OD600 reaches 0.2
3）By adding CYA to different sample tubes to a certain concentration, observing at a constant temperature 30°C for a certain period of time, the fluorescence intensity was measured by Thermo instrument (excitation wavelength is 503nm and the emission wavelength is 512nm), so as to explore the best induction condition and the response time range of the biosensor
4）Notes: during the pre-experiments, we notice that the LB medium had a serious background effect in measuring the fluorescence intensity, so all samples were measured after washing and resuspension.
Protein Extraction for Cell-Free Expression Reaction Use
*2X YT+P media preparation: 6 g tryptone, 10 g yeast extract, 5 g sodium chloride, 7 g potassium phosphate
dibasic, 3 g potassium phosphate monobasic, and water to one liter.
1. Inoculate 30ml initial bacteria solution into 250ml flask, add antibiotics and culture overnight.
2. OD600 was measured after 16h culture to confirm whether the growth was saturated. 2X YT + P medium was used as the blank control for measuring OD600 value. Then inoculate 20ml saturated fermentation medium into 1L growth medium to continue culture (2x YT + P needs to be preheated at 37°C 30mins in advance).
3. Culture for 3.5-5h at 37°C and 200 rpm until OD600 reaches 3. (after that, the solution, equipment and utensils used in the ice step need to be chilled to 4 degrees in advance)
4. Add 400 uL 1M DTT to the wash buffer to make its final concentration 2mM. Take two clean 50ml Falcon tubes, record the weight and keep them on ice.
5. Distribute the cell culture into two centrifuge tubes and balance them on ice—afterward, Centrifuge it at 4°C at 5000XG for 15 min. Carefully remove the supernatant and transfer the precipitate separately into a clean, 50mL Falcon tube. Select 25mL each of wash buffer previously mixed with DTT for resuspension, continue the process until no solid particles are present in the suspension
6. Centrifuge the solution at 4°C at 5000XG for 10 min, remove the supernatant and wash it with 25mL of wash buffer
7. Centrifuge, the solution at 4°C 5000XG for 10 min, remove the supernatant and wash it with 25mL of wash buffer
8. Centrifuge the solution at 4°C at 7000XG for 10 min, remove the supernatant, wipe all the exposed surface and the remaining fluid inside the tube. Weight the mass of precipitation.
9. Flash-freeze the cells in liquid nitrogen and store them under -80°C. Let the cells unfreeze on ice for one hour before attempting the next step.
10. Using Vortex oscillation, each gram of cell should be resuspended in 1mL of wash buffer.
11. Resuspend each pellet for 5 to 10 min before distributing 1.4mL of that suspension into four 1.7 mL Eppendorf tube.
12. Sonicate the suspension on ice. The optimal treatment capacity should be at 350J. After sonication, the suspension should have turned into a less sticky, brown liquid.
13. After lysis, add 4μL 1M DTT tp everu 1.7mL tube. Centrifuge the tube at 12,000XG, 4°C for 10 mins.
14. Pipette off the supernatants on top and combine them into two new Eppendorf tubes.
15. Cover the test tubes with aluminium foil and incubate them at 37°C, 200 rpm fro 80 min.
16. After incubation, the test tubes should turn opague. Centrifuge them at 12,000XG below 4°C and remove all unrelevant protein.
17. Add 600uL of 1M DTT into the dialysis buffer to stabilize its concentration at 1mM and transfer it into a beaker with a stirring rod. Soak the dialysis cassette (we used the Slide-A-Lyzer Dialysis Cassettes, 20.000 MWCO from Thermo Scientific) in the dialysis buffer for at least five minutes. Dry the cassette in a dark bix without contaminating the membrane.
18. After centrifugation, pipette off the supernatants and relocate the clear extract into the dialysis cassette.
19. 3 hours of dialysis should be conducted at 4 °C, timely change of buffer is not required.
20. After dialysis, remove the extract from the box and relocate them into two new, 1.7mL test tubes. Centrifuge the tubes at 2000XG 4 °C for 10 min. When we obtain the extracted product -protein, relocate the supernatant into a new testube and distribute it to multiple 35uL test tubes. Flash-freeze it on liquid nitrogen and store at -80 °C.
21. Determine the concentration of the extracted product.
1. Inoculate 30ml initial bacteria solution into 250ml flask, add antibiotics and culture overnight.
2. OD600 was measured after 16h culture to confirm whether the growth was saturated. 2X YT + P medium was used as the blank control for measuring OD600 value. Then inoculate 20ml saturated fermentation medium into 1L growth medium to continue culture (2x YT + P needs to be preheated at 37°C 30mins in advance).
3. Culture for 3.5-5h at 37°C and 200 rpm until OD600 reaches 3. (after that, the solution, equipment and utensils used in the ice step need to be chilled to 4 degrees in advance)
4. Add 400 uL 1M DTT to the wash buffer to make its final concentration 2mM. Take two clean 50ml Falcon tubes, record the weight and keep them on ice.
5. Distribute the cell culture into two centrifuge tubes and balance them on ice—afterward, Centrifuge it at 4°C at 5000XG for 15 min. Carefully remove the supernatant and transfer the precipitate separately into a clean, 50mL Falcon tube. Select 25mL each of wash buffer previously mixed with DTT for resuspension, continue the process until no solid particles are present in the suspension
6. Centrifuge the solution at 4°C at 5000XG for 10 min, remove the supernatant and wash it with 25mL of wash buffer
7. Centrifuge, the solution at 4°C 5000XG for 10 min, remove the supernatant and wash it with 25mL of wash buffer
8. Centrifuge the solution at 4°C at 7000XG for 10 min, remove the supernatant, wipe all the exposed surface and the remaining fluid inside the tube. Weight the mass of precipitation.
9. Flash-freeze the cells in liquid nitrogen and store them under -80°C. Let the cells unfreeze on ice for one hour before attempting the next step.
10. Using Vortex oscillation, each gram of cell should be resuspended in 1mL of wash buffer.
11. Resuspend each pellet for 5 to 10 min before distributing 1.4mL of that suspension into four 1.7 mL Eppendorf tube.
12. Sonicate the suspension on ice. The optimal treatment capacity should be at 350J. After sonication, the suspension should have turned into a less sticky, brown liquid.
13. After lysis, add 4μL 1M DTT tp everu 1.7mL tube. Centrifuge the tube at 12,000XG, 4°C for 10 mins.
14. Pipette off the supernatants on top and combine them into two new Eppendorf tubes.
15. Cover the test tubes with aluminium foil and incubate them at 37°C, 200 rpm fro 80 min.
16. After incubation, the test tubes should turn opague. Centrifuge them at 12,000XG below 4°C and remove all unrelevant protein.
17. Add 600uL of 1M DTT into the dialysis buffer to stabilize its concentration at 1mM and transfer it into a beaker with a stirring rod. Soak the dialysis cassette (we used the Slide-A-Lyzer Dialysis Cassettes, 20.000 MWCO from Thermo Scientific) in the dialysis buffer for at least five minutes. Dry the cassette in a dark bix without contaminating the membrane.
18. After centrifugation, pipette off the supernatants and relocate the clear extract into the dialysis cassette.
19. 3 hours of dialysis should be conducted at 4 °C, timely change of buffer is not required.
20. After dialysis, remove the extract from the box and relocate them into two new, 1.7mL test tubes. Centrifuge the tubes at 2000XG 4 °C for 10 min. When we obtain the extracted product -protein, relocate the supernatant into a new testube and distribute it to multiple 35uL test tubes. Flash-freeze it on liquid nitrogen and store at -80 °C.
21. Determine the concentration of the extracted product.