Team:Shanghai HS United/Experiments
| Buffer | 10 μL |
| dNTP. | 4 μL |
| Enzyme. | 1 μL |
| Template. | 5 μL |
| (PSB1K3-J23100-B0034-ter) | |
| Water. | 28 micro-liters |
| Primer1. | 1 μL |
| Primer2. | 1 μL |
| (PSB1K3-R) |
1.Add all the materials together:
•Firstly, use pipette to straw 28 μL pure water from the pure water bottle to one microcentrifuge tube.
•Secondly, use pipette to straw 10 μL buffer to the previous tube.
•Thirdly, use pipette to straw 4 μL dNTP to the previous tube.
•Next, use pipette to straw 5 μL template to the previous tube.
•Then, add 1 μL of each primer to the previous tube.
•Finally, use pipette to straw 1 μL of enzyme to the previous tube.
2.Place the tube to the PCR machine to react at a different temperature for each stage.
| NaCl. | 4 grams |
| Tryptone. | 5 grams |
| Yeast. | 2.5 grams |
| Water. | 500 milliliters |
1.Make the lysogeny broth (LB)
•Firstly, use balance to measure 4 gram of Sodium chloride and add to a conical flask.
•Secondly, use balance to measure 2.5 gram of yeast and add it to the conical flask.
•Thirdly, use balance to measure 5 grams of tryptone and add it to the conical flask.
•Use measuring cylinder to measure 500 milliliters of water and add to the conical flask. Stir the mixture in the conical flask with a glass rod.
2.Culture the bacteria with lysogeny broth (LB)
•Firstly, add 5 μL of antibiotic to the lysogeny broth (LB).
•Secondly, pour the LB liquid to a petri dish until the liquid fill the surface of the dish and cool them in the 37-degree-environment for 1 hour.
•Secondly, use pipette to straw 1micrometer of bacteria and add to the petri dish.
•Thirdly, use a clean plastic stick to spread the liquid on the original petri dish until the surface is completely smooth and even. Put the dishes in the 37-degree-environment for 24 hours.
3.Transformation of E. coli bacteria
•Firstly, use a pipette with a tube to closely touch the E. coli bacteria on the petri dish and mix it with plasmid in the tube.
•Secondly, put the tube into the ice for 20-30 minutes.
•Thirdly, put the tube back to the 42 degrees water bath for 90 seconds and ice them again for 2 minutes.
•Next, pour the lysogeny broth (LB) into the mixture in the tube and put it on the shaking machine for 45 minutes at 37 degree Celsius.
| Buffer. | 1.230 milliliters |
| Pure water. | 30 μL |
1.Firstly, straw all the liquid in the PCR tube except the precipitation we have used into a filter tube.
2.Secondly, use pipette with a tube to straw 130 μL of washing buffer to the filter tube which we have just used.
3.Next, put the filter tube into centrifuge and spin for 1 minutes.
4.Then, throw away the liquid in the bottom of the tube and add 700 μL of washing buffer to the filter tube.
5.Thirdly, put the filter tube into centrifuge and spin for 1 minutes. And add 400 μL of washing buffer to the filter tube again.
6.Fourthly, change the bottom tube into a new one while keep the filter.
7.Finally, add 30 μL of pure water at 65 degree Celsius to the new tube.
8.Lastly, put the filter tube into centrifuge and spin for 1 minutes and throw away the filter tube. The bottom tube is the recycle of DNA.
| Water. | 4.1 μL |
| DUTP. | 1.4 μL |
| Mixture. | 10 μL |
| (Buffer, DNTP, enzyme included) | |
| Template. | 2 μL |
| UDG. | 0.5 μL |
| Primer. | 2 μL |
| (Suggested using number 4 primer) |
1.Add all the materials together:
•Firstly, use pipette to straw 4.1 μL pure water from the pure water bottle to one tube.
•Secondly, use pipette to straw 1.4 μL dUTP to the previous tip.
•Thirdly, use pipette to straw 2 μL primer to the previous tube.
•Next, use pipette to straw 2 μL template to the previous tube.
•Then, add 0.5 μL UDG to the previous tube.
•Finally, use pipette to straw 10 μL of mixture to the previous tube.
2.Put all the tubes we have made into the water bath at 65 degrees for 30 minutes.
3.Do the following Cas12a procedure to detect if the solution includes the virus.
| Water. | 14.5 μL |
| Buffer. | 2 μL |
| Enzyme. | 0.5 μL |
| Template. | 0.5 μL |
| (From solution we made after LAMP) | |
| Detector. | 2 μL |
| crRNA. | 0.5 μL |
1.Add all the materials together:
•Firstly, use pipette to straw 14.5 μL pure water from the pure water bottle to one tube.
•Secondly, use pipette to straw 2 μL buffer to the previous tube.
•Thirdly, use pipette to straw 0.5 μL enzyme to the previous tube.
•Next, use pipette to straw 0.5 μL template to the previous tube.
•Then, add 0.5 μL crRNA to the previous tube.
•Finally, use pipette to straw 2 μL detector to the previous tube.
2.Straw 1 μL of the mixed solution in the tubes to the ELIASA Plate.
3.Detect the solution using ELIASA machine to check if the fluorescence value is within the range.
| Water. | 14.5 μL |
| Buffer. | 2 μL |
| Cas 12A Enzyme. | 0.5 μL |
| Template. | 0.5 μL |
| (From solution we made after LAMP) | |
| FITC Detector. | 2 μL |
| CRRNA. | 0.5 μL |
1.Add all the materials together:
•Firstly, use pipette to straw 14.5 μL pure water from the pure water bottle to one tube.
•Secondly, use pipette to straw 2 μL buffer to the previous tip.
•Thirdly, use pipette to straw 0.5 μL enzyme to the previous tube.
•Next, use pipette to straw 0.5 μL template to the previous tube.
•Then, add 0.5 μL CRRNA to the previous tube.
•Finally, use pipette to straw 2 μL FITC detector to the previous tube.
2.Put the solution into the environment of 37 degrees for 45 minutes.
3.Detect the solution using Cas12A test paper. If the solution contained the virus, there will be two red lines shown on the test paper；on the contrary, only one red line will be shown if the solution do not have the bacteria.
Materials:
| Washing Buffer 1. | 600 μL |
| Washing Buffer 2. | 700 μL |
| Pure water. | 60 μL |
| Ethanol | 250 μL |
| S1 | 250 μL |
| S3 | 350 μL |
1.Firstly, add 250 μL ethanol to the washing buffer 2 using pipette.
2.Secondly, use pipette with a tube to straw 250 μL of S1 to the tube contained the E. Coli bacteria.
3.Next, straw 350 μL of S3 using pipette and add it to the E. Coli tube.
4.Then, centrifuge using the machine for 10 mins.
5.Thirdly, straw the non-precipitation solution into a mini probe using pipette.
6.Fourthly, add 500 μL of W1 and centrifuge for 1mins.
7.Fifthly, add 700 μL of W2 and centrifuge for 1mins again.
8.After that, put the filter tube into centrifuge and spin for 1 minutes and throw away the bottom tube.
9.Then, add 60 μL of water at 65 degrees and wait for one minute.
10.Lastly, measure the concentration of the solution in the tube and mark the information on the tube for user in the future.
| Water. | 10 μL |
| DNA sector. | 1.5 μL |
| Template. | 5.5 μL |
| Buffer. | 2 μL |
| Enzyme Ezmax (Shanghai Tolobio company). | 1 μL |
1.Calculate the amount we require for DNA sector and template. As we need to ensure that the ratio for DNA sector and template is five to one and the total amount for the DNA sector and template is seven μL.
2.Add all the materials together: (this procedure should be done on ice)
•Firstly, use pipette to straw 10 μL pure water from the pure water bottle to one tube.
•Secondly, use pipette to straw 2 μL buffer to the previous tip.
•Thirdly, use pipette to straw 5.5 μL template to the previous tube.
•Next, use pipette to straw1.5 μL of DNA sector to the previous tube.
•Finally, use pipette to straw 1 μL of enzyme to the previous tube.
3.Put all the tubes we have made on ice for 30 minutes.
After the reaction, wait for 5 more minutes to stop the reaction and take it out.