Team:Nanjing/Notebook
Notebook
Hey guys! Please enjoy our process in the lab. It was really a meaningful experience, full of laugh and joy. Hope you enjoy our daily Diary!
Day 1:
Joyce, Justin, and I went to the lab today and we made the culture medium. Justin and I didn't use the pipette well at the beginning. There were always bubbles. We did better later but still a bit slow. I think we can be more practised next time.We started to prepare at around 4pm and began working on 4:30. Before 5pm, we weighed the mass of components in the culture medium and mix them with water. After 5:45, we did the sterilization for both the liquid and solid medium for 2 hours. The solid medium actually started to cool and solidify after we took it out. A kind of cloudy material at the bottom. We plan to transform cells tomorrow. Also, I checked the GOI we got. It's 1000ng, almost invisible. The company said we need to centrifuge the tube first because the powder may stick on the tube wall.
Day 2:
We transformed the E.coli DH5α today. During the process, we changed some part of the protocol. First, we reduced the amount of competent cell used in the experiment from 100μL to 5μL. The senior told us that our plan was for normal competent cells. However, the cells in the lab is of high quality, so we can reduce the amount. Accordingly, we reduced the culture medium amount added and skipped one step of centrifugation. We had culture plates. One is done by the senior with bacteria 200μL. I did the other one with bacteria 100μL because we didn't know which amount was the best. In case they grow too much tomorrow, we prepared two plates with different amount. After we pour the medium into the plate, we had 30 minutes of sterilization. It seems like a kind of ray, maybe UV light.
Day 3:
Here are our colonies! I put the culture dish of 100ul bacteria in the fridge as a backup plan. It has fewer colonies than the one done by the senior. We chose some single colonies on the solid culture medium and put them unto liquid medium. They need to shake for another night before we Can use their plasmids. It’s so exciting to see those small round spots growing on the medium. The seniors told me that it’s very likely to fail in the first time and I was not completely used to the apparatus yet. However, I did it! The colonies were absolutely normal, just like those on the senior’s dish.
Day 4:
We performed PCR of the GOI. Our protocol is slightly different from that on the instructions. We followed the instruction at last. I have recorded all the changes on the paper. It was a huge struggle for both the senior and me. The senior hadn’t performed PCR before and are not quite sure about the instructions. Luckily, the most experienced seniors (who the senior instructor of mine called Sister and Brother), taught us about the whole process. Interestingly, both the Sister and the Brother strongly blamed about how poor the lab condition and the machine was. The PCR machine was outdated and difficult to put the designed order in it. Finally, after a long discussion, we made an agreement. The GOI is put in the machine for a whole night, in the condition of 4 degree Celsius after replication finished. After that, we performed plasmid extraction, adding different solutions into bacteria and then we got the plasmids. The centrifuge roared when working. I worried whether it was okay to continue the process, but the seniors seemed used to the noise.
Day 5:
Taking the tiny PCR tube from the machine, we started to recycle the fragments in order for the next PCR. The main process is very similar to that of plasmid extraction, but a lot easier. I have to say the kit was really useful. We tested the concentration of DNA after recycle, on a huge machine which only use a tiny drop of DNA solution. The result was much lower than expected actually. Maybe we waste some gene fragments during recycle. Nevertheless, that didn’t matter too much because we still needed to perform another PCR. It was a mistake when we designed our recombinant plasmid. We add the restriction sites on GOI in opposite direction so we have to remove the original restriction sites first and then add another pair of sites in correct order. The new PCR process was of no difference to the previous one. How amazing the world of molecular biology that everything was actually changing while no one can see by eyes.
Day 6:
After recycling the PCR product again, we performed an electrophoresis to test whether the GOI and the plasmid was at about the correct length. We used 5μL system in order to save the materials. We made the agarose gel first. It was a very light powder, but when the solution was heated, it has a strong smell. After boiled, the senior cool it down a little bit and add florescent dye into it. She didn’t allow me to perform this because she said the dye is poisonous. We used five grooves —— one for GOI, three for plasmids, and one for marker. We pour TAE solution into the electrophoresis machine and then the gel, turning on power and wait for the result. However, after half an hour, we couldn’t see anything under the UV light. The Brother came and told us we have made a mistake on the direction. We put the grooves facing negative pole. So, we performed electrophoresis in the correct direction again and get our image. All the genes are in their expected position and are extremely clear. It means we can perform restriction tomorrow successfully.
Day 7:
We performed restriction today, still a not-too-difficult process with a long time of waiting. It was surprising that restriction was performed in PCR machine as well. The PCR machine was actually a thermal chamber and we can design the temperature changes. Like before, we couldn’t see what happened inside the machine. What we can do was simply praying that it can work.
Day 8:
Taking out the tube from the machine, it’s time for us to test our result. Another electrophoresis was performed, but we used 50μL system this time. The lab didn’t have such large model to make the grooves, so we stick two “broaches” together to make a larger one. We did the electrophoresis correctly this time. The image seemed to be good, and then we recycle the gel using gel recycling kit. The Brother helped us cut the gel with genes and put the bar into a centrifuge tube. I measured the weight. He but them so well that all the bars were exactly the same mass to the digit of 2. He didn’t even use any measuring tool during the process! The recycle was like PCR recycle, but a lot more complicated. Also, the plasmid is about 11kb. Although our kit showed it can recycle up to 40kb effectively, the senior told me it would still waste a lot of genes. After testing the concentration, the plasmid was only about 5ng/μL. The GOI was better. It was not a very satisfying concentration, but it still can be used to do ligation.
Day 9:
We did ligation today. Again, in the PCR machine. After calculating the amount of vectors and GOIs used in ligation, we put the tiny tube under 4 degree Celsius for the whole day. Tomorrow we will transform the new plasmids into bacteria again to see whether it worked.
Day 11:
It’s so sad we failed. No colonies were growing on the medium, which means the ligation didn’t work. It’s time for us to find out the problems during the process. We first checked the protocol to see whether there were errors. Interestingly, there wasn’t. We were confused. How can the result be like that? We thought maybe the concentration of vectors and GOI in ligation affected the process and we managed to increase the concentration and redo the whole process.
Day 13:
So sad we failed again, and it showed the concentration may not be the key factor. The Sister asked me to give her our primer design. The primers were designed by company Beyotime as sponsorship. Surprisingly, the Sister found that the error was in the primer design. The company didn’t add protective bases into the sequence, making PCR fail. We contacted the company immediately and asked them to synthesize the primers again. It will take about five days for the new primers to arrive.
Day 19:
Our new primer arrived and it was time for us to repeat almost all the process. I hope we can catch up with the time.