Day 11 - Science Quiz by the Prof and Aquarium Cleaning session

Day 11 - Science Quiz by the Prof and Aquarium Cleaning

Today is a very tiring day although I did not have many things to do in the morning. The only thing that I did in the morning was a midi-prep. This time Ni already warned me many times not to make mistake since I did not managed to isolate any plasmid for my last Midi. Shen Qian and Foong Mei were only watching as they have never done this before. I made lots of mistake like pouring the LB into the water container that Ni uses for dispensing pipette tips instead of pouring it to the beaker... Sigh... But in the end I still managed to isolate the plasmids....

In the afternoon, after lunch, Prof Hong decided to test our knowledge (or our memorizing skill about restriction sites). So we went to his office and he asked us to write the restriction sites that he asked us to memorize last week on his white board. He told us to think fast and treat it as if we were having a science quiz where we have to buzz as quick as possible. To make the quiz even more interesting, he decided to give us some incentive which is new tissue paper to wipe the white board (Swt... -___-") if we could get 100% correct sites. So if we could not answer them correctly, we have to use the used tissue paper or wipe the board with our hands (-__-")

After the quiz, we have to go to the fish tank room to clean the fish tank. It was a very tiring job. First, we have to transfer the fish from the small tanks into big tanks. Then we have remove all the dirt from the bottom of the tanks and the sponge. Third, we have to adjust the water pipes so that the water would be dripping instead of spurting. Then we remove half of the water from the tanks (the tanks are fixed to the racks, so we cannot just pour the water out) before putting some salt to the tank. The problem was there are hundreds of small tanks in the room and we have to clean them all. In the end, I got a blister on my finger because the tap of the pipes were new and they were very tight.

Day 10 - the Witch of the fume hood

Day 10 - the Witch of the fume hood

That day, Wang Li taught us how to make LB plate for growing bacteria. There are so many autoclaving (sterilization) steps involved to ensure that the plates are not contaminated by bacteria.

We started with weighing the agar and the LB powder, followed by addition of RO water and autoclave inside the machine. After that, we had to pour the LB plate solution to the petridish inside a fume hood that had been sterilized using UV light for 15 minutes to kill all the bacteria.

When we came to the fume hood, we saw that the UV light was already switched on, so we thought that someone may has forgotten to switch it off. Since the fume hood was shared by the whole lab, we just thought that we can use it and that's what we did. We just switched the UV light off and add Ampicillin to the solution and start pouring it to the petridishes.

Suddenly, a skinny middle-aged women appeared behind us and without any hesitation she just said in a nagging voice "Do you know that you can let the agar solidified outside the fumehood?" In other words, she was trying to get us out of the fumehood. In the end, we did not finish making the agar plate because of her.

I asked Ni whether that fume hood belong to a specific professor and Ni said it was shared for the whole lab. Wang Li told us that she was always there everyday before 5, that's why she thought that the fume hood belongs to her... What an irritating old egoistic witch she was... Sigh

Day 9 - New Face in the Lab

Day 9 - New Face in the Lab

There was a new girl that would be joining our Lab for her Honour Project. Her name is Foong Mei and she came from Malaysia. Because she was new, Shen Qian and I explained to her what we were doing in the past few days. We started with our daily routine, PCR from the fin samples.

Day 8 - competent cells prepared by incompetent Scientists

Day 8 - competent cells prepared by incompetent Scientists

The morning was a bit relaxed as we only need to do PCR. Just put the PCR mixtures in the machine and wait for them to be done. Like usual, I used the waiting time to learn about Chinese as I left my Chinese text book in the lab. However, I didn't get any band for the PCR result. I guess I have either loss the enzyme or I loss the template... Sigh... I needed to redo it again....

The chapter that I learned for my Chinese lesson was about birthday so I announced my birthday to everyone in Chinese. My birthday is in August and unlike everyone, I hated my birthday. The incident that happened on my 18th birthday was still fresh in my mind. The feeling of being alone, dejected and self pity on that day had created a trauma on me. It was because none of my friends bothered about my birthday on that year hurt me so much. Wang Li suddenly said that he would organize an outing on my birthday.

In the afternoon, we learned about how to prepare competent cell for bacteria transformation. We just follow the steps and aliquot the cells which are ready to use into small eppendorf tubes and immediately freeze them by throwing it into liquid nitrogen. Every time we throw the tubes into the liquid nitrogen, we would heard a fizzle sound just like if we were frying something. The bacteria inside the tubes were immediately frozen and the liquid nitrogen emitted white vapor just like dry ice.

Day 7 - Lab's inspection

Day 7 - Lab's inspection

In the morning, we continued with our plasmid isolation and proceed to restriction digest (RE digest). Basically what we do in RE digest is to digest a recombinant plasmid with some enzymes to confirm the presence of DNA insert in the plasmid. However, I forgot to label my tubes and Rei Juan took them out... Sigh.... So, my plasmids were not digested properly.... I re-did my RE digest in the end...

Ni also asked us to redo our PCR until we could get reproducible results. We had to be able to get the same result 3 times. We did PCR twice with the same samples today and both of them look different... Sigh....

During lunch, Ni suddenly became very excited and start talking about her past idols such as Britney Spears. She then started singing "Baby One More Time" which make Shen Qian and I frozen in terror. It was in the canteen during lunch hour... Many people will look at us and thinking that we were too stressed (which may be true)...

While we were autoclaving the pipette tips, a plump lady came around and started to talk in Chinese with Ming you. Shen Qian whispered to me that she was inspecting our lab. Based on Shen Qian translation, that plump woman were telling Mingyou that he was not supposed to put any personal belongings on his table such as his laptop (which was always there) and his place was too messy. Shen Qian also told me that she heard from someone that the inspector dislike Ni because she always wear skirt in lab (we are supposed to wear long pants and covered shoes in lab). Luckily Ni was not around as she went somewhere else and she was wearing a long flowing skirt today. But this time her skirt was longer and she wear a covered shoes instead of her usual pink look-like-Croc sandals.

Day 6 - Chinese Lesson in the Lab

Day 6 - Chinese Lesson in the Lab

When we came to the lab, Ni showed us her new PCR result. This time she didn't dilute the sample and she had very nice bands. So, she concluded that there are not many DNA in the sample, hence there was no need for dilution and she asked us to redo our PCR.

While waiting for our PCR to be finished, Ni showed us how to isolate plasmid from the bacteria cells. She expected us to do the same after she finished hers. Actually both Shen Qian and I had done this before but we couldn't recall every single steps so we just watched how Ni did her mini-preps.

Since we incubate our inoculation in 30 degree (the incubator for 37 degree is broken and has not been repaired), we need to wait for longer time for the bacteria cells to be ready for plasmid isolation. While waiting, we went to student lounge to do our own stuff. I brought my Chinese text book with me and started reading while Shen Qian was reading her German. Wang Li corrected my prounounciation. I still could not believe it that he is only 1 year older than me and he is currently doing his PhD... I feel so left behind... Sigh....

After about an hour, we started our mini-preps. I stopped in the middle of my preparation as I need to leave for RA meeting. I froze my pellet while Shen Qian continues with her preparation. After I went back from my meeting, I continue my plasmid isolation but I didn't have enough time to finish it. So, in the end, I just stopped at the incubation step and helped them to autoclave (sterilize the laboratory equipment) pipette tips. Sometimes Shen Qian played a prank with Dr Li Mingyou by pasting a tape with "I am a bad guy (in Chinese character)" on his back.

At around 6 pm, Shen Qian, Mingyou and I went for dinner. During the dinner, we talked about some of the people in the lab who had obtained their PR then Mingyou mentioned about his intention of buying HDB flat but he couldn't since he's still single. We then persuaded him to find a girlfriend but he said that he's too busy that he didn't have time to get to know girls. He sounded very desperate that day... Sigh.... I am wondering whether I would be like him in the future, a desperate PhD holder....

Day 5 - What's wrong with PCR today?

Day 5 - What's wrong with PCR today?

The first thing that we did in the morning when we arrived in the lab was to run a gel for our PCR (genotyping) result. We did not get the band that we wanted. Instead we got a single band for each lanes on uniform size, the size of the primer dimer, which means that our PCR result is not successful. Thus, Hong Ni asked us to redo our PCR.

We repeated every step, but this time we made our own PCR master mix, and run a gel again. At the end of the experiment, I still got primer dimer but Shen Qian got a band at 1 kb. However, Shen Qian result was still not acceptable as we supposed to get at least some lanes with two bands.
Hong Ni decided that she would do the PCR to check the quality of the sample.

Today we also inoculated some bacteria. The bacteria is E.coli that had been transformed with plasmid vector with an insert. Each of us inoculated 8 colonies. After we finished with inoculation, Hong Ni briefed us on what is the schedule for tomorrow. But while she was explaining to us on what we were supposed to do on the next day, prof Hong came. Instead you were wondering, Hong Ni is professor daughter and she is currently doing her PhD in her father's lab.

He then told us that we were not supposed to be so attached to Hong Ni as she has her own project. Prof Hong then brought us to his office and tell us what we were supposed to be doing and gave us some home works. We were supposed to memorize at least 10 different restriction enzymes, their sizes, recognition sites and buffers. He also told us to practice opening and closing the 1.5 ml eppendorf tube with one hand. After that, we went to the shaker where we incubate our inoculation tubes as we thought that we need to loosen the cap.

At the shaker, Prof Hong found some inoculation tubes that were two third filled with LB medium. Luckily those were not belong to us. We are supposed to fill one fifth of the tubes as the bacteria needs air to grow.

After we had our dinner, we went back to the lab to see Hong Ni's PCR result but she also only get primer dimer bands. So, we were wondering what actually went wrong.

Day 4 - Butches among fish?

Day 4 - Butches among fish?

My mentor told me that we couldn't do Western Blot today because we could not get fish embryo to do it. He just asked me to follow whatever Shen Qian and Hong Ni were doing. So I went to their bench to join them.

The schedule for that day was to do RNA isolation and Genotyping the fishes. The purpose of Genotyping is to determined whether the fish is genotypically male. The difference between female and male fish in medaka is at their back fin. Male medakas have discontinuous jagged back fins whereas their female counterparts have continuous smooth back fin. However, not all medaka with discontinuous jagged back fins are males, some of them are genotypically females (so I called them butches, female with male appearance).

After preparing our RNA sample from medaka's ovary and ran them for RT-PCR, we went to the aquariums to get some supposedly-male medakas to be genotyped. What we did was to cut a small part of their tails, homogenize it to extract the DNA, perform PCR and run gel electrophoresis. If the fish is genotypically male, two bands will be observed from the gel run, but if the fish is actually a female fish, only one band will be observed. We took samples from 34 fishes and put them back in separated aquarium.

After that, we went back to our lab to homogenize the sample using a pestle. The pestle really looks like a large size cotton bud. It is white in colour and has two bulging ends. Crushing 34 small pieces of fish tails were quite tiring, luckily we split our jobs. After we homogenized the sample, we added celects, a reagent that will bind to everything that will interfere with PCR. Then we performed PCR to each sample.

While waiting for PCR to finish, we run a gel to visualize our RNA isolation sample. The result was very disappointing as I only can see a smear on my lane showing that my RNA has been completely degraded. Most probably I was not careful enough while handling the reagents... Sigh....

In the end, we decided to run our gel for genotyping on the next day as it was getting late.

Day 3 - Lab meeting (Saturday, 16 May 2009)

Day 3 - Lab meeting (Saturday, 16 May 2009)

On the third day, we had a lab meeting at S2 building. This was my first lab meeting. When I arrived there, I could not enter the room as I did not have an access, luckily a nice gentleman helped me to open the door. I came too early and apparently only Dr. Xu was there.

When everyone was ready, Prof Hong introduced Shen Jien (a Honour Student) and I (the UROPS student) to the whole team as we were still new. After we have one round of introduction, he told everyone about the new water circulation system in our fish tanks and persuade them to speak english all the time. Well, all of them except for me came from China and they speak Chinese most of the time.

The speakers for the day was Yanyan and Zhendong. Yanyan talked about gene targetting of p53 gene by homologous recombination in medaka ES cell. By the end of his presentation, he also mentioned that he wanted to do a further experiment in combination of p53 and NOS3 genes but Prof Hong discouraged him as both gene used the same anti-biotic resistant. Thus, the selection of the successful tranformed cells will not be effective. Zhen Dong talked about nanod (a gene that maintain prulipotency of the cell) in medaka and zebrafish. Prof Hong commented about how he present the data as well as the needs for statistical evidence.

I left in the middle of the meeting as that day I have to attend RA training but I did learn something about data presentation.

Lab day 2

Day 2 - Friday, May 15, 2009

Today, I did micro injection again. This time, I injected GFP (Green Florescence Protein which make the embryo glow in the dark) for some of the embryo and concentrated Phenol Red (which only stains the yolk red for temporary moment for training purposes). I collected the fish roe myself. This time I managed to inject them successfully although I was pretty sure that some of the embryo were destroyed again. But it was pretty cool to see the ball of cells glow in the dark, hehehehe.....

Three to Four hours after the injection, I learned how to separate the cell mass from the yolk. It was very very difficult. I had to break the corion first to release the embryo and then separating the cell mass from the yolk but every time I break the corion, the yolk and the cell mass were squizzed out of the egg and were mixed together. In the end, I only managed to isolate half of the cell mass from eight embryos. The rest of the embryos were destroyed in faint. I guess I have to practiced more.

Yesterday embryos has developed well although some of them were dead. I could see the embryo squirming under the microscope.

After that, I supposed to learn how to do Western Blot, a technique to detect a specific protein. But my mentor was engrossed in dissecting his fish and by the time he finish, we didn't have enough time to do Western Blot. I ended up joining another group on doing PCR... Sigh.... At this present moment I still have not started my project yet....

Lab day 1 - Thursday, May 14, 2009

This was my first day doing UROPS (Undergraduate Research Opportunity Programme is Science). My mentor, Li Zhendong, is a PhD student from China. I've met him 2 days ago and he is a very nice person. He is currently busy with his paper so he doesn't have much time to teach me.

On the first day, he introduce me on how to do micro injection. It is a technique used to introduce a substance to a cell through injection. In my case, it's injecting RNA or Morpholino (a substance which block mRNA from being translated into protein) to Zebrafish's egg yolks.

The technique was not as easy as I thought it would be. I have to prepare my own needle for the injection and ensure that I only inject the substance once, not twice, to every embryo. The machine was controlled with a pedal and for a person who are not used to pedalling (like me.... I haven't passed my driving test yet due to bad pedalling), most of the times the substance was injected twice.

At the end of my first training day, I ended up wasting 5 needles and destroyed some of the embryo. Some of the air bubbles were trapped inside of the needles, making them useless or sometimes I made a blunt opening of the needle... Sigh....

That day, I also revised PCR technique and have smudged bands... T.T.. sigh....