Protocol

1.Running PCR for pLKO.1-pur plasmid

Aim of the experiment: PCR stands for polymerase chain reaction. In essence, it is a vitro replication of template DNA. Using a simulated environment for the Taq polymerase to work. It aims to get more available gene for further experiments.

Mix approach:

1. Melt the frozen fluid with ice, which is roughly 4 degrees, wait for roughly 20-30min (fluid must be melted thoroughly)

2. Make sure that the fluids are in correct concentration (most of the time, plasmid and primers require dilution)

25μl Mix solution(xTransStart" FastPfu Fly PCR SuperMix)

2μl forward primer (pLKO.1-puro f)

2μl reversed primer (pLKO.1-puro r)

25μl Mix solution(xTransStart" FastPfu Fly PCR SuperMix) 2μl forward primer (pLKO.1-puro f) 2μl reversed primer (pLKO.1-puro r)

20μl ddH2O

3. Add the solutions (2xTransStart" FastPfu Fly PCR SuperMix, pLKO.1-pur, forward and reversed primer) in ratio based on the provided ratio on the instructions paper, into a suitable Ep tube

4. mix the fluid thoroughly and make sure to cool fluids down to roughly 4° Celsius while waiting for other Ep tubes to be mixed

5. place the Ep tubes into the PCR machine.

6. Create a new session and set the pre-denaturation, denaturation, annealing, extension, post-extension, and post-PCR temperature. (For the mix xTransStart" FastPfu Fly PCR SuperMix its accordingly: 98°,98°,60°,72°,72°,4°) Also make sure the timing of the different procedures is correctly set (labeled on instruction paper) (1min, 10sec, 5sec, 10sec,1min, ∞). And command to repeat the denaturation, annealing, extension process roughly 30-35 times. (Additional info: the time of the final extension step of the main cycle may differ from the chosen mix, it may differ from 10sec to 1min)

7.start the PCR machine accordingly

8. wait roughly for 50-60min

2.Experiment and results of gel electrophoresis for reacted pLKO.1-pur plasmid

Main aim: The main aim of gel electrophoresis is to test the length and size of the DNA or RNA, to check if our previous work is correctly inserted (such as PCR).

1.First of all, we need to make the gel. All the water that is involved in gel electrophoresis is a solution of water with TAE buffer, which maintains the pH to about 8.3 to keep DNA/RNA/plasmid to flow fluently in the gel. Instruction and recipe are taken from iGem official (50X TAE BUFFER PREPARATION (100 mL)).

“Tris Base 24.2 g
Glacial acetic acid 5.71 ml
EDTA (0.5 M) 10 ml”

Then we mix roughly 1-2% of agarose according to the mixture amount, for a 100ml gel, we use roughly 1g of agarose powder. Mix thoroughly.

2.Then, for the agarose gel to mix and form a liquid fluid, we need to put it in the oven and heat it until there can be visible bubbles forming on top of the solution (roughly 70-80°) (normally the homogenous solution melts at roughly 60°C).

3.Add TAE buffer into the mixed and cooled agarose gel. (ratio and amount) (name incorrect)

4.Set up the mold for the gel. Normally we use a standardized mold and choose our “comb” according to the amount of gene material that we choose to add.

5.Then we wait for the solution to cool down to a suitable temperature for the mold.

6.Pull out the comb after the gel solidifies

7.Additional biomarker is added in the template DNA, and then is moved to each of the hole. For our experiment, 10μl of DNA is mixed with 2μl of DNA loading buffer, which works together to provide the illuminance when we view the results.

8.Place the gel into the middle of an electrophoresis tank already filled with TAE water. Make sure that the TAE water soaks the gel fully. (Check: make sure that the direction of the gel is correct, where the long side/running side faces the positive electrode (normally red), and the short side faces the negative electrode (normally black))

9.Move the DNA material into the holes of the gel.

10.Add the biomarker to the most right or left hole for control compare

11.Plug in the electric wire onto the socket above the electrode

12.Open the electrophoresis machine, and adjust the voltage and current, and time. Normally, the voltage is roughly 120V, for the sake of time, we set the voltage to 130V.

13.Run the machine and wait for roughly 30min

14.After the waiting. If you view the gel again there will be signs of “running”.

15.To view the results, the gel must be placed in a special UV machine that captures the iridescence.

16.Compare the results to the control biomarker, and evaluate weather if the PCR was successful.

3. Obtain the recombinant sh-TEAD4 plasmids

Main aim: Normally this experiment is conducted with the aim of replicating a reconstructed plasmid, so normally after this experiment, the plasmid will be implanted into eukaryotic cells for the expression of the reconstructed plasmid. However, we just did a demonstration of the process of implanting a plasmid into E. coli and culturing the bacteria. (normally this experiment is for replicating the edited template plasmid, while we just use an unmodified plasmid) (PCR does achieve the same results, but the length of the bacterial plasmid is significantly longer than the length of our template DNA even if it's in the form of plasmid (template DNA: ~≤2000, bacterial plasmid: normally 5000~≤) )

Making LA:

1.First, we need to make a LA culture media. 10g/L tryptone (0.5g/50ml)
5g/L Yeast extract (0.25g/50ml)
10g/L sodium chloride (0.5g/50ml)

To make LA we add a 15g/L of agar sugar. In our case, we add 0.75g of agar sugar

2. After we mix the solution thoroughly (use ultrasound mixing if agar sugar isn’t fully dissolved), we need to sterilize the solution. Specifically, we put it in a high- pressure sterilizer for about 30min, under 121°C and 0.1Mpsi (remember: use a selectively permeable membrane that will only allow gas to flow freely).

3.After waiting for the sterilization process, wait for the solution to cool down to roughly 50°C to ensure that the AMP+ (ampicillin) won't denature.

4.The AMP+ used is 1000 × so 1ml of LA contains 1μl of AMP+. So, when we resize the AMP+ amount, we need 50μl of AMP+ in each 50ml of LA.

5.After the AMP+ is added to ensure that other bacteria don’t interfere its growth, the LA must be quickly poured into a petri dish or else it will start to solidify. The process is done in a clean bench.

Reconstruction of E. coli:

1.The core idea of reengineering E. coli is to insert a plasmid that contains an inserted template gene (ask: what criteria will we need to choose a plasmid that minimalize other expression). So, the E. coli must be in a natural competence state, which gives its characteristic of diffusing a whole plasmid through its peptidoglycan cell wall. For us to do this, we need to make a special order of E. coli cells that is in its natural competence state.

2.The next thing to do generally is to create the reconstructed plasmid. (we didn't do an experiment on this) First, we prepare the original plasmid (add: criteria of choosing) that we want to modify.

3.Find a suitable endonuclease (suitable here means that there isn't any repeating sequence both in the plasmid and in the template DNA)

4.Design a primer for the template DNA, and put a random codon sequence in front of the endonuclease sequence the primer for protection purposes (remember: there are a forward and reverse primer, which represents both sides of the DNA).

5. Do the PCR experiment according to the description above using the designed primer.

6.The preparation of a sliced plasmid includes endonuclease and a chosen plasmid. Normally the endonuclease works at roughly 37 degrees. (details about this experiment)

7.After the slicing, run a gel electrophoresis to the separated plasmid.

8.The results are a long DNA (the one that we are going to use) separated with a shorter DNA (the one that we are going to replace)

9.Recycle the longer sequence gel (do gel recycle)

10.(ligase reaction)

11.To implant the plasmid into the bacteria, we need to further more excite the bacteria, and implant the plasmid during this process. So, we inject our modified plasmid into the Ep tube that contains the E. coli.

12.First, we put the Ep tube in ice for about 30min to fully melt it.

13.Then place the Ep tube into a 42°C water bath for 45sec.

14.Place the Ep tube in ice again for 2-3min again. (Through this process, the plasmid enters the E. coli)

15.Now, move to the clean bench and prepare the culturation of E. coli on LA

16.Basically, we just need to inject 10μl of modified E. coli onto the LA and lay the Bacteria flat on top the LA using a glass rod.

17.Now place the LA into a 37°C incubator for 12-15h

18.After the incubation, prepare single clone selection.

Single clone selection:

1.Check if the LA culture dish is cultured with bacteria. Normally, a successful E. coli culturation is when there are just white dots and without too much odor. This is signaling the use of AMP+.

2.Move everything into a clean bench, including a jar of LB

3.Take a glass bottle, with suitable cap

4.Inject 2ml of LB into the bottle

5.With the ratio of 1000ml LA/1ml AMP+, add 2μl of AMP+

6.Then use the thinnest possible pipette gun head or a tooth pick to pick a bacterium with a single clone (this ensures the replicated bacteria is pure, and is specific)

7.Inject the whole pipette gun head or drop the tooth pick into the 2ml of LB

8.Close the cap, and put it on a Labatory shaker with 220rpm for 12-15h, set temperature to 37°C.

4. Plasmid extraction

Main aim: After culturing the

1. Extract 1ml~5ml of cultured bacteria in the form of LB and place it into a suitable Ep tube

2.Centrifuge the Ep tube in 10000 ×g for 1min

3.Remove the centrifuged liquid, and make sure that there is bacteria separated underneath

4.Add 250μl of solution I/ RNase A and mix thoroughly, make sure all of the separated bacteria are fully mixed.

5.Add 350μl of solution II and mix gentle until there is a condensation of cotton like DNA in the solution

6.Centrifuge the Ep tube in 13000 ×g for 10min

7.Move the upper liquid into a mini column and throw away the separated rest of the EP tube.

8.Centrifuge the tube for max g-force (13000 ×g~16000 ×g) for 1 min

9.Remove the additional centrifuged liquid below

10.Add 500μl diluted HBC buffer (diluted with ethanol) and centrifuge with max g-force for 1min.

11.Add 700μl of DNA wash buffer and centrifuge with max g-force for 1min.

12.After removing the waste fluid, put the mini column back into the centrifuge and centrifuge the Ep tubes in max speed for 2 min to fully remove any kind of buffer.

13.Add 15-30μl of elution buffer onto the mini column gently and centrifuge the Ep tube at 13000 ×g for 1 min to wash out the DNA.

14.Remove the mini column and store the DNA in the –20-degree refrigerator.

5. Plasmid transfections

Main aim: to store cells for a long time, we normally put the cells in liquid nitrogen tanks, cell recovery is when we want to use the preserved cell again. We aim to not damage too much the cell because of the heat change.

1.To follow the rules, once you enter the cell incubation room, you need to make sure that you aren't contaminated with bacteria, this is because that cells can be easily affected by bacteria.

2.Label the date and time you took away the bacteria on the general notebook for reference

3.Different cells are required to use different culture medium, and CRC cells that we are using requires to use L15 complete medium, which is a mix of L15 culture mediumand fetal bovine serum in the ratio of 9:1.

4.After taking out the frozen cell from the liquid nitrogen, put it in a 37°C water bath.

5.After the cell is fully recovered pour all of the cell into 4-5ml of L15 complete medium

6.Place the mixed cells into the 5% CO2 incubator, and wait for 12-15h for incubation

6. CCK8 experiment

Main aim: the main aim of cell counting board is to quantitatively count cells. It can be used to compare the differences for pre-drug and post-drug cell proliferation ability. Count the cells in the small squares, sum it up together, divide them by four and times it by 10^6 for the number of cells in 1mL fluid.

1.Prepare the cell counting board, make sure its washed clean fully

2.Prepare a microscope slide, make sure that it covers four of the squares on the counting board

3.Bring the cell culturation fluid and the counting board into the clean bench.

4.Add 10μLof cell fluid onto the counting board .

5.Due to adhesion, the fluid will totally spread throughout the board .

6.Put it under the microscope and follow the counting process.

Demonstration of CCK8 (cell counting kit) using CRC:

1.First, we want to separate the cell from the plate so we can dilute it and plant it into the 96 well plate. So, we add buffers to the plate and wash off the cells, then we centrifuge them and add L15 complete medium to dilute the concentration of the cell.

2.Add equal amounts of cell fluid to planed holes in the 96 well plate. Normally, plant 100 μL f fluid into each hole to maintain roughly 3000-10000 cells per well.

3.Incubate overnight (24h).

4.Remove the previous culture medium (remember to wash with PBS) and add new culture medium that contains 10% of CCK8 diluted fluid. (throughout this process you don't want any bubbles to form within the culture dish to affect the measure outcome)

5.Incubate for another 0.5-4 hours.

6.Put theplate into a Fluorescence Reader.

7. Detection of ROS level

Main aim: ROS is a type of chemicals that exist naturally within a human's body. Normally there is a redox balance of ROS with other substance. However, in cell pathology, excessive ROS can cause cell apoptosis, and additional ROS may lead to more proliferation, which is related to tumorigenesis. DCFH-DA is the dye that we are using, DCFH-DA can diffuse through the cell membrane and be reacted into DCFH, when there is excessive ROS, it will reduce DCFH into DCF, and DCF is illuminance. We can indicate the ROS level when we measure the intensity of illuminance.

1.Separate the cell from the culturation dish. Add buffers to the culturation dish and wash off the cells, then we centrifuge it and dilute the concentration of the cell.

2.Separate the culturation fluid into the six holes with roughly 3-5*10^6 cells in each, incubate for 24 h .

3.After incubation, remove culturation medium and wash with PBS twice.

4.After washing the board, add a DCFH-DA solution that is in the ratio of 1:1000 to L15 medium, make sure that it covers all cells

5.Incubate for 20 min.

6.Wash the non-diffused DCFH-DA thoroughly in a non-illuminated environment .

7.Put it in a fluorescence microscope and observe and take pictures.

Transwell experiment to test the migration ability of tumor cells:

8. Transwell experiment

Main aim: the main aim of transwell is to test the migration ability of the cells. There are micropores through the membrane that the tumor cells move through. Once we add fetal bovine serum to the other side, because of the attraction of nutrients, the tumor cells migrate to the side with more nutrient. After the migration, we count the amount of tumor cells that has migrated.

1.Add 500μl of culture medium with fetal bovine serum into the 24-hole board.

2.Put the plate nto the hole filled with the serum (8μm pore)

3.Put the cultured cells into each of the “well”, which is 200μL of fluid containing 2*10^5 cells

4.Incubate for 20-30 h.

5.Take out the plate and place it into a crystal violet solution and incubate in an no light condition for 15 min

6.Gently soak the dyed “well” in water until all free dyes are washed out

7.Use a wet cue tip to remove the upper layer of cells and wait until its dry

8.Put it under a microscope and take pictures for counting the cells.

Final words: and that's all the experiments we have done throughout this amazing trip! These are definitely useful!!!