When I made a mistake, my PI never let it go. It would be mentioned again and again.
When he made a mistake, he shrugged it off, saying everyone made mistakes and should move on.
The part that he admitted him made a mistake was not a part of everyday life, but the part he accused me of doing sth wrong is the part of everyday life.
Monday, August 26, 2019
Friday, August 23, 2019
100 ways for experiments to go wrong in a biology lab
I start this log because there are so many things happened in my lab that I am not even sure I should cry or laugh. Thus, I decided to start writing down stories that happened in this lab so that the readers can judge themselves.
Chapter Li, Winter, 2018
I was trying to reproduce an immunostaining image in the literature.
My PI asked me to try some random dye to stain the nuclear of a cell, rather than using DAPI, a well-established reagent. I asked why I could not use DAPI, he commented on he didn't wish to waste money on another laser line.
I quietly followed his protocol. Initially, staining nuclear with ToPro3, which fluorescence could be collected with 647 channel, worked.
Then I started to see weird patterns on my images, and eventually, being able to collect no good data.
I mentioned this problem to my PI. He said I must have done something wrong, and I should simply repeat everything again with his watchful eyes.
I tried. Then the weird pattern on the image attracted his attention.
It turned out that the filter for the fluorescence collection was burnt. Thus it was normal that I could not get anything useful.
Chapter Li, Winter, 2019
A more detailed version can be found here. In short, my PI asked me to purify a protein. I failed 3 times, and every time he accused me of having bad hands.
It later turned out to be he gave me the wrong protocol. He mixed up the formula between elution buffer and wash buffer. For the whole time, I had been, under his instruction and supervision, using elution buffer to wash my beads for purification.
Chapter Milda, Spring, 2019.
Milda was trying to re-produce a published result, which is YAP, a protein, is localized in the cell nuclear when the cells are on a stiff matrix. Yet she saw no signal in her immunofluorescence image.
My PI started with speculating Mida did something wrong, and then speculated the antibody might not be the best. But nothing seemed to be the cause. He then asked Dr. Rodionov, a person who has decades of experience in immunofluorescence and extremely nice, to help Milda to troubleshoot.
Dr. Rodionov could not find any apparent issue. However, he suggested that Milda could use his more expensive formaldehyde, rather than the cheap substitute in my lab.
And then everything worked. There was bright immunofluorescence of YAP, overlapping with Milda's DAPI stain for the cell nucleus (DAPI).
Chapter Prem, Summer, 2019
Prem is interested in regulated secretion. He is using fluorescent protein-tagged insulin to gauge the process of insulin secretion, and other proteins that may be involved in it.
In the literature, people used C-terminus tagged yellow fluorescent protein (mVenus, a bright red-shifted variant of GFP) to achieve this goal.
In his case, he used a C-peptide tagged human insulin. Previous research suggested that this is a better fluorescent protein tag than the C-terminus tagged. They are more likely to be folded correctly.
Previous in house data suggests this C-peptide tagged insulin goes to granule like structure and can be trafficked in the MIN6 cells, a pancreatic beta cell line with mouse origin. This insulin can be secreted out of MIN6 cells when the cells are stimulated with glucose, like real beta cells. This was shown in both literature using biochemical data and in-house with live-cell imaging.
Prem wished to use total internal reflection fluorescence microscopy to gauge insulin secretion. Thus, he tried to switch the fluorescent protein tag from GFP to pHluorin. pHluorin is a pH-sensitive variant of GFP. In the acidic environment, pHluorin is dim; in neutral pH, the fluorescence becomes brighter. Secretory granules are acidic, while the extracellular medium has a neutral pH. Thus, it was believed that when the MIN6 cells are stimulated with insulin, he should be able to see lots of bright puffs that signals insulin secretion.
However, in the real experiment, he saw nothing.
Later research suggests that pHluorin might go through a different post-translational process (i.e. different glycosylation). This may be the reason why he cannot see anything in his experiment.
Chapter Li, Summer 2019
I have been trying to capture insulin receptor A, an isoform of the insulin receptor, enrichment in primary cilium in MIN6 cells. I was able to capture ciliary enrichment of human insulin receptor A with interdomain GFP tag using immunofluorescence.
However, I was unable to achieve the same thing in my live-cell imaging.
My PI thinks this is due to the GFP is too dim so there isn't enough signal for me to capture. Thus, he forced me to switch the GFP tag into a more brighter fluorescent protein, called mNeonGreen.
Yet still. I see no IR-A-mNeonGreen in the primary cilium.
Then I tried to immunostain the cells to see whether IR-A-mNeonGreen goes into the primary cilium anyway. To my surprise, I cannot see the receptor's ciliary localization.
Then I ran a western blot, comparing IR-A-GFP and IR-A-mNeonGreen expression in cells. Oddly, despite mNeonGreen is only 3 amino acids difference from GFP, there is a significant molecular weight shift in the mNeonGreen tagged receptor.
I guess this is another "post-translational modification messed up my experiment"!
I hate all these fluorescence protein manufacture not to put these on the warning.
What a waste of time, my youth and energy.
Chapter Li, Winter 2019
I was trying to repeat some experiments I did in the past. This is an experiment to test whether our design of photo-activatable insulin receptor (optoIR) can be activated by light. If our design works, we are expected to see light-induced receptor phosphorylation.
We were able to see light-induced receptor phosphorylation for a couple of times. But later we cannot repeat the experiment after we start to illuminate our sample from the top through the lid, rather than from the side.
We later found out that we cannot see light-induced phosphorylation because of water droplet condensation on the top of the lid. The condensation blocks the light thus we see no light-induced receptor phosphorylation.
Chapter Li, Winter 2020
I was trying to repeat an experiment I did before. Previously, when I stimulate my cells with this stimulus, I was able to see fluorescence change from the cells that express a fluorescence-based biosensor (fluorescent protein) on our microscope. The experiment outcome has always been repeatable.
But somehow I didn't see this happen in a January day.
It turned out that my PI changed the recipe of our imaging medium.
Everything worked again when I changed the medium back.
Chapter Li, Winter, 2018
I was trying to reproduce an immunostaining image in the literature.
My PI asked me to try some random dye to stain the nuclear of a cell, rather than using DAPI, a well-established reagent. I asked why I could not use DAPI, he commented on he didn't wish to waste money on another laser line.
I quietly followed his protocol. Initially, staining nuclear with ToPro3, which fluorescence could be collected with 647 channel, worked.
Then I started to see weird patterns on my images, and eventually, being able to collect no good data.
I mentioned this problem to my PI. He said I must have done something wrong, and I should simply repeat everything again with his watchful eyes.
I tried. Then the weird pattern on the image attracted his attention.
It turned out that the filter for the fluorescence collection was burnt. Thus it was normal that I could not get anything useful.
Chapter Li, Winter, 2019
A more detailed version can be found here. In short, my PI asked me to purify a protein. I failed 3 times, and every time he accused me of having bad hands.
It later turned out to be he gave me the wrong protocol. He mixed up the formula between elution buffer and wash buffer. For the whole time, I had been, under his instruction and supervision, using elution buffer to wash my beads for purification.
Chapter Milda, Spring, 2019.
Milda was trying to re-produce a published result, which is YAP, a protein, is localized in the cell nuclear when the cells are on a stiff matrix. Yet she saw no signal in her immunofluorescence image.
My PI started with speculating Mida did something wrong, and then speculated the antibody might not be the best. But nothing seemed to be the cause. He then asked Dr. Rodionov, a person who has decades of experience in immunofluorescence and extremely nice, to help Milda to troubleshoot.
Dr. Rodionov could not find any apparent issue. However, he suggested that Milda could use his more expensive formaldehyde, rather than the cheap substitute in my lab.
And then everything worked. There was bright immunofluorescence of YAP, overlapping with Milda's DAPI stain for the cell nucleus (DAPI).
Chapter Prem, Summer, 2019
Prem is interested in regulated secretion. He is using fluorescent protein-tagged insulin to gauge the process of insulin secretion, and other proteins that may be involved in it.
In the literature, people used C-terminus tagged yellow fluorescent protein (mVenus, a bright red-shifted variant of GFP) to achieve this goal.
In his case, he used a C-peptide tagged human insulin. Previous research suggested that this is a better fluorescent protein tag than the C-terminus tagged. They are more likely to be folded correctly.
Previous in house data suggests this C-peptide tagged insulin goes to granule like structure and can be trafficked in the MIN6 cells, a pancreatic beta cell line with mouse origin. This insulin can be secreted out of MIN6 cells when the cells are stimulated with glucose, like real beta cells. This was shown in both literature using biochemical data and in-house with live-cell imaging.
Prem wished to use total internal reflection fluorescence microscopy to gauge insulin secretion. Thus, he tried to switch the fluorescent protein tag from GFP to pHluorin. pHluorin is a pH-sensitive variant of GFP. In the acidic environment, pHluorin is dim; in neutral pH, the fluorescence becomes brighter. Secretory granules are acidic, while the extracellular medium has a neutral pH. Thus, it was believed that when the MIN6 cells are stimulated with insulin, he should be able to see lots of bright puffs that signals insulin secretion.
However, in the real experiment, he saw nothing.
Later research suggests that pHluorin might go through a different post-translational process (i.e. different glycosylation). This may be the reason why he cannot see anything in his experiment.
Chapter Li, Summer 2019
I have been trying to capture insulin receptor A, an isoform of the insulin receptor, enrichment in primary cilium in MIN6 cells. I was able to capture ciliary enrichment of human insulin receptor A with interdomain GFP tag using immunofluorescence.
However, I was unable to achieve the same thing in my live-cell imaging.
My PI thinks this is due to the GFP is too dim so there isn't enough signal for me to capture. Thus, he forced me to switch the GFP tag into a more brighter fluorescent protein, called mNeonGreen.
Yet still. I see no IR-A-mNeonGreen in the primary cilium.
Then I tried to immunostain the cells to see whether IR-A-mNeonGreen goes into the primary cilium anyway. To my surprise, I cannot see the receptor's ciliary localization.
Then I ran a western blot, comparing IR-A-GFP and IR-A-mNeonGreen expression in cells. Oddly, despite mNeonGreen is only 3 amino acids difference from GFP, there is a significant molecular weight shift in the mNeonGreen tagged receptor.
I guess this is another "post-translational modification messed up my experiment"!
I hate all these fluorescence protein manufacture not to put these on the warning.
What a waste of time, my youth and energy.
Chapter Li, Winter 2019
I was trying to repeat some experiments I did in the past. This is an experiment to test whether our design of photo-activatable insulin receptor (optoIR) can be activated by light. If our design works, we are expected to see light-induced receptor phosphorylation.
We were able to see light-induced receptor phosphorylation for a couple of times. But later we cannot repeat the experiment after we start to illuminate our sample from the top through the lid, rather than from the side.
We later found out that we cannot see light-induced phosphorylation because of water droplet condensation on the top of the lid. The condensation blocks the light thus we see no light-induced receptor phosphorylation.
Chapter Li, Winter 2020
I was trying to repeat an experiment I did before. Previously, when I stimulate my cells with this stimulus, I was able to see fluorescence change from the cells that express a fluorescence-based biosensor (fluorescent protein) on our microscope. The experiment outcome has always been repeatable.
But somehow I didn't see this happen in a January day.
It turned out that my PI changed the recipe of our imaging medium.
Everything worked again when I changed the medium back.
Post candidacy: the tale of protein purification
Last night I shared my experience in protein purification with my roommate, who is specialized in studying protein. She laughed so hard that I think I should write down my story, which took place this February.
My PI asked me to purify a protein, a FRET sensor with HIS tag at the N teminus. He gave me a protocol out of his head, claiming this was what he had been doing for a decade and this is the protocol that worked for the previous graduate student, Abhi.
He asked me to start by preparing the bacteria soup in the volume of 100ml. Yet I can see no fluorescence in the tube after elution. He accused me of having bad hands so that I must have lost all the proteins during purification.
He then asked me to prepare the E.coli soup in 1 litre so that I could have more room to make mistake yet still yield purified protein. The bacteria soup and all these reducing agents smelled so terribly I had to wear a plague mask, but even that I still suffered from a terrible headache induced by the stench. Yet nothing worked.
He then accused me of not following the protocol religiously and asked me to have him watch me when I was doing my experiment.
I did it again. I showed him the bright yellow fluorescence (from the YFP in the FRET sensor) in the bacteria pallet yet nothing yield in my elution solution.
It was the day of recruitment. I remembered potential candidates walked in the lab, and I was on my plague mask. I had to apologize for the stench.
Later after dinner with candidates, he approached me and told me that he made a mistake. He mixed up the formula between elution buffer and wash buffer.
For the whole time, I had been, under his instruction and supervision, using elution buffer to wash my beads for purification.
I guess it is always a great story to tell.
My PI asked me to purify a protein, a FRET sensor with HIS tag at the N teminus. He gave me a protocol out of his head, claiming this was what he had been doing for a decade and this is the protocol that worked for the previous graduate student, Abhi.
He asked me to start by preparing the bacteria soup in the volume of 100ml. Yet I can see no fluorescence in the tube after elution. He accused me of having bad hands so that I must have lost all the proteins during purification.
He then asked me to prepare the E.coli soup in 1 litre so that I could have more room to make mistake yet still yield purified protein. The bacteria soup and all these reducing agents smelled so terribly I had to wear a plague mask, but even that I still suffered from a terrible headache induced by the stench. Yet nothing worked.
He then accused me of not following the protocol religiously and asked me to have him watch me when I was doing my experiment.
I did it again. I showed him the bright yellow fluorescence (from the YFP in the FRET sensor) in the bacteria pallet yet nothing yield in my elution solution.
It was the day of recruitment. I remembered potential candidates walked in the lab, and I was on my plague mask. I had to apologize for the stench.
Later after dinner with candidates, he approached me and told me that he made a mistake. He mixed up the formula between elution buffer and wash buffer.
For the whole time, I had been, under his instruction and supervision, using elution buffer to wash my beads for purification.
I guess it is always a great story to tell.
Friday, June 7, 2019
PhD post candidacy: mourning
It has been two month since the death of my beloved Sangria. Legally she is my property. Traditionally people don’t mourn for the their dead pet publicly. Yet I find it hard to get over the grief. She was one of the best things happened in my PhD years. She helped me calming my nerve, release my stress, and give me an excuse to rest at home. Without her, I am drown in work, and I feel my emotion and mental status are at the brink of collapse.
Yet my work keeps me so busy that I barely get a minute off to write a proper eulogy for her. Every falling flowers keeps reminding of the ending of her life. I am not sure whether it is the pollen or her keeps me crying.
I could never commemorate her too much.
鷓鴣天Sangria
不解街邊貓沈思，未讀國風嫌惡詞。灰鬢不度香腮雪，偷嗅鏡中影是誰。 柳絮嫁，杜宇離，鶯愁蝶倦晚芳時。只怕惜花心易碎，青冢不見荼蘼枝。
Yet my work keeps me so busy that I barely get a minute off to write a proper eulogy for her. Every falling flowers keeps reminding of the ending of her life. I am not sure whether it is the pollen or her keeps me crying.
I could never commemorate her too much.
鷓鴣天Sangria
不解街邊貓沈思，未讀國風嫌惡詞。灰鬢不度香腮雪，偷嗅鏡中影是誰。 柳絮嫁，杜宇離，鶯愁蝶倦晚芳時。只怕惜花心易碎，青冢不見荼蘼枝。
Sunday, June 2, 2019
Post candidacy: PhD is a scam
I realized something funny when I was talking to my boyfriend.
About two years ago, I was preparing for a journal club. In that paper, the authors used both mouse and rat models. I asked my colleague, a person that hold PhD from a German institute, what the difference is between mouse and rat. He shrugged, and replied he wasn’t sure. He then commented “I think there is a difference, but I don’t know. It’s difficult to tell since English is not my mother tongue “.
About two years ago, I was preparing for a journal club. In that paper, the authors used both mouse and rat models. I asked my colleague, a person that hold PhD from a German institute, what the difference is between mouse and rat. He shrugged, and replied he wasn’t sure. He then commented “I think there is a difference, but I don’t know. It’s difficult to tell since English is not my mother tongue “.
Friday, May 24, 2019
Thursday, April 11, 2019
Cherry Blossom 西江月 樱
The whole Ph.D thing has never been easy for me. Ever since I started to do experiments, I gradually lose my mind. I guess I am constantly stressed out. So far, nothing has worked yet. I hate my lab for every ill in my experiment. My PI's constant scolding and belittling only make me bitter.
A blossom from the cherry blossom, or sakura, gives me little comfort. I guess I am happy the minute I left Connecticut for DC. I am yet more depressed on my way back.
Yet life needs to go on. And life sucks.
I am only praying for my experiments to work so that I can see the light of graduation.
Saturday, March 2, 2019
obituary: Margarita the rat
When I look back at this photo that is taken on Jan 16, 2019, I wonder whether Sangria knew Margarita was dying.
I wish she could tell me what happened. But I guess I would prefer she was unaware of any abnormality if ignorance could spare her pain.
When I was preparing for my journal club, Margarita slept soundly on the butt of Sangria. I called her name, she didn't bother to open her eyes. Instead, she turned her butt towards me, as if I was a bad smell she wanted to aviod. I thought she was bored with endless scientific talking took place in my room. I never anticipated that would be tha last time I saw her alive. I probably bored her to death (literally) with my journal club.
Margarita the rat was born in the forest of Farmington in my friend's place. She and her siblings are from an accidental preganancy. It was not known to my friend that their mother was pregant. They arrived to this world anyway.
I adopted Margarita, as well as her sister, Sangria, from my friend, and brought them home, a quiet neighborhood in West Hartford. My landlord's dog didn't seem to be interested in these two little furry balls. From early age, Margarita was the beta rat. She was suspicious about everything around, like hiding in the dark, and being very cautious when I approached them. In contrast is her sister, Sangria, a very outgoing and a very curious, and dominant rat. It didn't take long for Sangria started to bully Margarita, occasionally beating her despite being very close to her for the rest of the time. They sometimes groomed each other, slept on each other, fought for the precedent in taking food, and whom to occupied the better box or hammock.
There were early signs that Margarita is not the dominant, the more outgoing rat. Shortly after they arrived my home, Sangria started to explore the cage, while Margarita stayed at the shadowy corner of the cage. It was Sangria who started to take treats from my hand. For some time, Sangria was the only rat took things from me, while Margarita was standing aside watching Sangria. Not until she was sure that there were no danger to her would she join Sangria to take treats. Yet there were still significant difference between these two eating: while Sangria would stay near by my hand so that she could get her next piece of cereal (cereal was the treat) as soon as she finished last one, Margarita preferred to take the treat and run to a corner of the cage in the shadow and finish her food there. I was slightly upset by Margarita's suspicion, as if I were some threat to her. I need to admit that I may not be completely friendly to her. They annoyed me when they made their cage completely a mess and when they peed everywhere on my table. I sometimes scolded them loudly, hoping this is a type of discipline so that they would not do it again. Instead, this only made them more timid, especially true for Margarita. I guess she could not understand what human means. Maybe in her eyes, I was a giant that that makes noise all the time, despite this level of noise will attrack preditors, such as cats. This put all of us in danger.
I have to admit that I take them as members of my household not fully due to the kindness out of my heart. I was worried that I may develop allergy over time because living condition in this country is very clean. On the other hand, I was also lonely. I had to endure a boyfriend that was never around, barely cared for what I need, and didn't put up the effort to read what I enjoyed. I was very sad when I heard one of my friend's boyfriend learnt Python just to understand what she was going through, and how ridiculous Python could be. I hope some furry balls to keep me and my immunosystem occupied so that I would not feel lonely or develop allergy.
My rats never failed that part to entertain my immunosystem. They never cease to make their cage a complete mess, or making my room full of dust (from their bedding) and the smell of rats. They endless pee on my tables and my sheets so that I have to keep cleaning all the time. Overtime, they also develop a habit of tearing my sheets/ pillows/ pillow cases/ shirts so that they could take some pieces of fabric home and made a nest for themselves. Sangria, as the more outgoing rat, had been the one to initiate this vandalism. Margarita, with endless energy, always carried out what Sangria started.
It took sometime for Margarita to get use to live in my household. During the early days, Margarita always hid in the shadow of Sangria. Sometimes she didn't seem to be bother by the fact that Sangria used her as a pillow or a cushion. She sometimes fought Sangria, but only end up to be beaten. There were a couple of times I heard Margarita screaming after Sangria beating her. It didn't bothered Sangria that she hit Margarita's face to get herself in the front line to get cereal. Nor did she show any level of remorse when she took cookies from Margarita by force. Margarita, tried her best to defend her cookie in vain. She hit Sangria's face hard, trying to send Sangria a message to stay away from her cookie. Nevertheless, Sangria has stronger teeth and limbs and Margarita ended up standing with no cookie.
There is one task, however, Margarita out-performed Sangria: nesting. Margarita loves to make nests from paper/ tissue/ fabric and boxes. She and Sangria together has torn apart 4 sheets, a comforter, a pillow, and endless pillow cases so that they can use the fabric/ cotton to make their own next. Margarita is particullay fond of tissue boxes. She viewed these boxes as perfect starting point of making herself a home. She was very persistant when making nest. She would repeat the action again and again and again until she had her nest. In constast, Sangria's interest in nesting was short-lived. She would be interested in taking fabric home for about 2 or 3 minutes, before starting to wondering around and find new interesting things.
I guess Sangria saw no necessity in making a nest. If she wanted one, she would take a nest from Margarita. Once I added a new tissue box in their cage, adjacent to the tissue box that is already in their cage. Margarita had converted the old tissue box there into a comfortable nest with all the pieces of paper and fabric she stole from me, many of which with scientific material. Neither of them seemed to be interested in the newer tissue box. Sangria, initially running around the cage, decided it was high time for her to go home. Yet she didn't want to reside in new box. Instead, she crowded into the old tissue box, the box that Margarita "decorated" and was sleeping in. Initially, Margarita didn't seem to care about sharing the box with her sister. However, maybe 5 minutes later, the two rats started fighting, and Margarita was kicked out the nest she made. She was so helpless standing at the entrance of the box, wishing to go back to the nest she built for herself. Yet Sangria blocked her way, declaring the box was hers now. Margarita ended up converting the new tissue box into her new nest.
Yet this episode does not mean that Sangria does not nest. Only she nested differently. She enjoyed taking pedals from my flowers and use these pedals for her nest, while eating anthers for food. I once joked to Sangria that a hundred-year-old person said his secret for longevity is to eat a tulip everyday. The next day, Sangria ate my tulip. I guess she wanted to live for 3 years so that she can greet people new year at the year of rat.
Atlas, Margarita is not there to join her.
Yet started 2019, I noticed that Margarita became much more active than she used to be. In comparison, Sangria spent more time stayed inside the cage doing nothing. Margarita were more likely to respond to the sound of cereal and jumped out of cage for the treats. She also seems to be timid than she used to be. She was rather relaxed when I was around, enjoying herself. I thought this was a good sign: after years hidden in the shadow, Margarita was willing to take a more active role in my household.
I was preparing my journal club the other night. I was under a huge amount of stress, as I knew all the professors there would be very sharp, and they had a record of asking difficult questions. It didn't help when a friend sent me a screenshot showing my journal club was at "UConn Today" page. This meant there would be more outsiders in my journal club. Who knows what they would ask. I was rehearsing again and again, and Margarita seemed to be very bored by it. I tried to pursuade her to learn something about receptor trafficking in primary cilia, but she seemed to couldn't care less, and was asleep. I guess she understood well that she did not have ciliopathy so that she could not care how other people has messed up primary cilia. Being bored of me talking to her, she turned her butt to me tp show that she was not interested in it at all.
Yet when I waked up the second day and boiled two eggs for them as breakfast, I only see a restless Sangria. I went year the cage and see Sangria was standing on Margarita. I didn't think much of it, and tried to wake Margarita up for her breakfast. Yet I found she was cold. I was in shock. I fell on the floor, keep patting her, wanting to see her looking at me inpatiently. Yet she didn't move. She lied still, with her eye half open, yet she didn't respond.
I could not morn more about Margarita. She didn’t outlive her mother. I blamed her early death on Sangria, for her endless bullying may cause chronicle stress on Margarita. I cannot cease to blame myself as well, since my presence may also cause stress on my very sensitive Margarita. I wish she died in peace, and had little regrets about her life.
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