the unluckiest day

The unlukiest day...

I was very busy (or should I say lazy to update this blog) these few days...

To summarize everything:

Day 17 - repeat

When I did sequencing, ZD was not there. So I asked Wang Li and he told me that I only need to do 2 out of 10 samples so I only did 2. When ZD came back he told me that I need to do all 10 because this is the first sequencing as no one ever try to clone the gene before so I have to repeat the whole procedures for 8 more samples. At the end of the day I need to prepare bacteria culture for Midi preps the next day.

Day 18 - Linearization

After the midi-prep, I did a test digestion to determine the orientation of the insert.
Since I am going to do in situ hybridization, I need to linearize the plasmid that are going to be used as a template for constructing the probe. It's basically only digestion with enzymes. I just left those in the incubator overnight

Lab meeting - weekends

The presenter was Wang Li and dr Yi. But they were talking about our Lab secret experiments so I could not reveal those in this blog. After the meeting, I went back to the lab to take out the linearized plasmid and then help Ni to fill in tips. She paid me 10 cents for every box....

Day 19 - running gel

ZD asked me to run gel to determine whether the plasmid had been completely linearized. Unfortunately only 1 has been linearized. ZD asked me to make another linearization and somehow after 3 hours, I still get the same result... So in the end, I resort to gel extraction. I just load all my samples to the gel and run the gel for about 1 hours. After that I cut the gel and freeze it as I had no time to extract the gel

Day 20 - the unluckiest day

After gel extraction, ZD told me to purified the gel. However, he forgot to tell me that I should use DEPC water instead instead of TE buffer as the template need to be RNAse free. So he told me I need to repeat everything from linearization. Luckily I still have some plasmids left.

Today, SQ brought the pillow that I asked her to buy from IKEA. I had been suffering from a severe pain on the upper part of my thigh everyday after lab as a result for sitting for long hours. Then ZD told me that pillows are forbidden in the lab to avoid contamination as the fabric is absobant and hard to clean. So I wrapped the pillow in thin polyethene layer. Everyone was just giggle to see the pillow deflatten and made a farting noise everytime I sat on it.

Day 16

Day 16 - Lucky

It was a busy day after inoculation

I had to do mini-preps followed by test digestions. I did two test digestion: the first one using EcoRI and the second one using BamHI and XhoI. My first test digestion did not give good result as I did not get the band that I want. ZD said that probably I must redo the experiment from ligation step. I was so scared. Two days would be wasted.

But later, when he re-examined my test digestion result, he said that it seems that only the test digestion was problematic so I only required to repeat the test digestion. Luckily the second test digestion came out fine and all lanes showed the band that is required. ZD said that I was so lucky. The next day, I just need to do sequencing.

Day 15 - "I don't care, throw it away"

Day 15 - "I don't care, throw it away"

There was nothing much to do on that day.

I checked my plate in the morning and there were some colonies growing. However, the colonies were too small so I could not see the color. I have to wait for a few more hours before the colonies are big enough for me to see their color.

After about 3 hours or so, I went back to check and they are ready to be harvested. So I went to the fumehood to do inoculation. However, ZD saw at the LB medium that he gave me the day before. The LB was murky, showing some bacteria growth, which means that I contaminated it somehow. So in the end, I have to ask ZQ and FM for some LB... Sigh... ZD even asked me to measure the OD value of that contaminated LB to show me that the LB has been contaminated.

I had to wait for at least 20 hours before I could proceed with my experiment. So I just hanged around in the lab. The other thing I did was only taking picture of the fish embryo under the microscope. SQ and FM was doing RACE-PCR (Rapid Amplification of cDNA 5'-end) and ZD told me that I could follow what they were doing if I want to but he "don't care". That day I realized that "I don't care" is ZD's favorite and overused phrase. During lunch we were talking about ZD and Yovita told me that ZD's previous favorite phrase during her UROPS time was "What is that?! Throw it away!" I told them that I was wondering how does ZD's girlfriend felt if he had one.

So when we went back to the lab I asked ZD, "Do you have a boyfriend?"... Yeah, I wrongly said that and everyone who heard me laughed, including ZD. He said "It's a secret" then I told everyone, the girlfriend must have felt very sad because I can imagine few scenarios.

Scenario 1
The girlfriend baked a cake for him and he said, "What is that?! Throw it away!"

Scenario 2
The girlfriend bought a new dress, purposely dress up for him and ask for his opion and he said, "I don't care"

Scenario 3 (The worst scenario)
The girlfriend told him, "I am pregnant with yourchild" and he said "I don't care. Throw it away!" OMG....

Day 14

Day 14 - Panic

In the morning, I showed my PCR result to ZD and he just said "Congratulation". So after that, I proceed with TA cloning. TA cloning is the whole process of cloning DNA using the advantage of the overhang A that was produced by the taq polymerase during the PCR reaction. In the TA cloning, the insert (PCR product) has overhang A while the vector contains an overhang T. Thus, they can form complementary base pair which make it easier for ligation.

At first, I misinterpret ZD instruction. I purified my PCR product without leaving any for TA cloning. In the middle of purification process, suddenly ZD just told me that if the purification process was not successful, I have to repeat the PCR. I felt so stupid and so stressed because of that. Luckily the gel run showed that my purification process was successful... Fiuh....

For ligation, instead of using ligase, we use topoisomerase. I don't really understand the whole process but it's obviously much shorter and simpler than using ligase. After ligation, we proceed with transformation but I had to wait until ZD finished preparing the competent cell. He told me that the competent cell that SQ, FM and I prepared last time was contaminated and "not good". So he prepared new competent cells.

After transformation, I just spread the transformed bacteria into a LB plate with IPTG and wait until the next day to select the colonies that has been successfully transformed.

Weekend

Weekend

It's weekend and yet we still have to go back to lab....

Well, if you noticed that I actually never wrote anything on weekend unless I have anything to do in lab.

When I went to the lab and checked the PCR machine I was shocked. The machine was turned off and it was opened. I could not find our (Shen Qian's, Foong Mei's and mine) PCR results and I was panicking (you could see that from my FB profile on that day). Luckily Shen Qian and Foong Mei came and they looked for it. Someone actually put it in the fridge.. LOL...

So we run the gel and Taa Daa... We got our bands. Mine and Foong Mei's were successful but Shen Qian did not add template into her tubes so she did not get the bands. I went back to PGP while SQ went to library while waiting for her repeat PCR to be done

Day 13

Day 13 - Let the project begin

When Zhen Dong came to the lab in the morning, he asked me where were the embryos. I told them I only had 10 (which were the remaining embryos that I had not given to Shen Qian and Foong Mei) and he sort of scolded me. The previous day he told me to collect at least 20. Then, I told him that I had isolate some RNA from lots of embryo yesterday. So I just pray that my RNA had a decent quality.

I waited for Shen Qian and Foong Mei before I ran my gel to see the RNA bands. And once again, I made a mistake. I loaded DNA ladder instead of RNA ladder but fortunately, the result was good. At least I got 2 clear band and 1 fuzzy band. the 2 clear band corresponded to rRNA while the fuzzy band at the lower part of the gel correspond to the tRNA. The smear at the band was the mRNA.

After that, we proceed with reverse transcription-PCR (RT-PCR) to isolate and amplify the desired mRNA. In RT-PCR, complementary DNA (cDNA) was produced using RNA as a template, therefore the name Reverse Transcription. After RT-PCR, we performed PCR using the primer that Zhen Dong and I have designed (the sample) and Beta-actin primer as our positive control. We left our PCR in the machine overnight as we were short of time.

Day 12 - the Hairy Roes

Day 12 - the Hairy Roes

That day I finally had a very vague idea of what my UROPS project is about

I am doing a research on a gene called VOX and to be honest, I don't have any idea of that gene except that the gene is expressed on stage 13 onwards in the fish embryo. Zhen Dong promised to send me a paper regarding VOX gene but until now I have not received it.

The first thing I have to do was to confirm the sequence of the VOX gene and to do that, there are multiple steps involved. The very first thing I did was to select the Medaka embryo which were already at stage 13 and above.

Medaka roes are very different from Zebrafish roes. While zebrafish's are smooth, medaka roes are hairy. The roes are clustered together with a very fine strands of brown fibre. Although the fibres were very fine and thin, they were very strong. The fastest way to separate them is to wind them around a pair of needle.

After selecting the embryos, I proceed to do a RNA isolation. I did not know what was wrong with me that day, maybe I was just to nervous to start my project, that I made lots of mistake. I forgot to aliquot the trizol reagent to smaller container and I even dropped my tip inside the bottle. I could sense that Yanyan who were there at that time was thinking that I was just a nuisance in the lab.

Foong Mei and Shen Qian had nothing to do so they wanted to join me in my experiment. I gave them some of my fish embryos. I guessed they were too lazy to select the embryo as they just took a bunch of them to the tube.

I did not finish my RNA isolation so I stopped at the washing steps.