nothing's done

It's been busy these few days but I felt like I've done nothing. Lab notebook is now a few pages thicker than Friday but there are no long paragraphs written in green ink, which is the color I use to write discussions and progress. There are tons of paragraphs written in blue ink, which is the color I use to write why the result came out that way and what could have gone wrong. There are just too many ways things could have gone wrong. So what's wrong?

When the PCR worked, I was overjoyed. I tested 20 µl reactions for several temperatures and I decided that it worked best at 65^oC. So I decided to increase the reaction volume to get more products -- 100 µl. However, I did not multiply the DNA polymerase by 5 because I felt that it was too much polymerase and I can’t afford to use that much since 20 µl of polymerase costs USD22. For each reaction, I would require 1.5 µl polymerase if I were to multiply by 5. That means one tube of polymerase would not even allow me to run 20 reactions…too freaking expensive. No wonder Invitrogen can still afford to hire during the recession period in US. Unfortunately, the lesser amount of polymerase I used gave me a different kind of product, one which I don’t need at all. So now I have to change the conditions to get the product I want and this is taking a whole lot more time. There’s only 1 week and 4 days left to ASM. The fear of not being able to finish is eating me up.

While I'm running all these manipulations, I'm not progressing because I have yet to get my product. Sad. It really does feel like nothing has been done. Need to hurry..

~worried~

simple and fun

On Friday, my plan for this weekend was to watch DVDs, sleep in and wake up without an alarm clock, read novels and read some articles on microbial diversity. However, I could not sleep past 9am even though it was dead quiet when I woke up except for the sound of the cars passing by on the highway once in a while. But that wasn’t the source of my waking up. It was more due to the habit of waking up at 6am every morning and all I need was a couple of hours extra. But I got the pleasure of just lazing around the house, watching Hero and Jersey Girl.

Unfortunately, it is impossible to leave lab work even for a day so I went to lab in the late afternoon to run a PCR. Yeap, the PCR is working well but I still can’t get the product I want to amplify in large amount. It seems like the primers have another annealing side that keeps giving me an unwanted product that is only about 800bp. But I am glad that I have products to play around with instead of just empty reaction tubes with just buffer and water.

Around 6pm, CY called and said that they are having a barbeque at Hawk Island County Park. What else would be more fun on a Sat. evening than spending time with friends by the lake, eating BBQ chicken drumstick? So I agreed to it and he came to pick me up. I am glad that I went because we had so much fun and it’s been such a long time since I laughed so hard and it was genuine. I didn’t have to pretend to be a different person. The gathering was nice too because there was no gossips, which would have occurred if I had spent time with M’sians from my undergrad peers. I am not trying to offend anyone but everytime there is a gathering, there’s always stories going around about who’s doing what and what is happening to who. Here, we don’t know each other very well except that we’re all M’sians and we appreciate companionship of our own kind once in a while. So it was just enjoying the moment and nothing more.

After the BBQ, we had a few games of bowling at Okemos and pool at Owen Hall. I came home late and did not get to read much stuff but it was fun. This is actually the first time I went out with them as friends and not because it is organized as an M’sian Student Organization activity. I would definitely do it again.

line

I've been chatting to BK quite a bit these days. He broke up with his gal and he's heartbroken about it although he's handling it well. We don't usually chat much about he and his gal but more on general stuff..what's going on..teasing each other..just casual chat. But sometimes the topic would go to his relationship with his gal and how he's trying not to think about it. I listen and soothe him with encouraging words. Am I affected? Well, at least I'm not longer crying over it and losing my mind but it still hurts. It hurts because he's telling me how much he's affected by his break up and yet, when we broke up, it was nothing to him..at least that's the impression I got. So should I listen and bare with the hurt or should I just ask him to shut up? Where do I draw the line? As a friend, I don't want to see him get hurt and I don't mind being there for him..but hurting myself? Is that worthit? I guess I kinda know the answer to all this question in my heart but I just had to let it out. I hope he'll have an easier time letting go of her..surprisingly, not because he would be available again but because I don't want to see him get hurt.

Thankfully the experiment worked today. At least I have something to rejoice about instead of being miserable over my uncontrolled feelings.

water!!!

For the past two weeks, I've been working on amplifying a gene encoding three proteins fused together - yellow fluorescent protein:beta-galactosidase:cyan fluorescent protein - by PCR. Unfortunately, all I've been able to get were empty gels when I ran the PCR product on the gel. I've manipulated several conditions and components of the reaction and still came up with nothing.

Yesterday, Dr. R suggested that I try using the DNA Grade water from Fisher for the reaction or get a new batch of dNTP mix from Invitrogen. I did both but to amplify the 16S rDNA gene from Verrucomicrobium, which is a positive control and guess what..the reaction with the DNA Grade water worked!!!!

Just before I left work today, I started the reaction to amplify the yfp:lac:cyp gene, which was my initial objective. Will only get to see if it works tomorrow but I have a good feeling that it'll work. Can you believe that something as simple as water can screw up experiments for two weeks??? I autoclaved my water for 30 minutes and aliquoted it out into sterilized Eppendorf tubes and never did open it again until I need to use it. So the only possibility that's screwing up the PCR is probably some mineral residues in the bottle during washing or there is an inhibitor in the bottle and it was not washed thoroughly.

I can't wait till tomorrow to check it but I'm soooooo glad I find out what's wrong with the reaction for positive control.

~ecstatic~

Blood Red Tulips

Blood Red Tulips, originally uploaded by lilsQuirr3l.

First time I've seen this color of tulips, my favorite color too. This was at the Tulip Festival at Holland, MI. It's about an hour west of Lansing and the Tulip Festival is to celebrate the time when all the tulips first start to bloom. This was at a small garden and there's more pics taken from the Tulip Farm. Wanted to buy some but completely forgotten about it when we were about to leave. It is such a beautiful flowers and the variety of colors, I never thought it's possible to have so many types of varieties for a particular flower species.

weekend

Went to Ohio this weekend with Nok. The main purpose was to attend Ben’s convocation and Ben had free meals at a really classy Japanese restaurant. She told us that we had to be in Cleveland by 6pm. We got there a little before 6pm and called her. She was on her way back from Kent. So we waited in the car for about an hour and she called back, saying that she had already moved the reservations for the restaurant from 7pm to 8pm. That made Nok a little angry because she doesn’t like to wait around for people even if it’s her sister. Furthermore, when Ben said the original time she booked was 7pm, it frustrated her even more because we were not informed of that either. At that time, Ben was already close to Cleveland but Nok was so angry that she didn’t want to attend the dinner anymore. So we went to an Asian store at downtown Cleveland, which was about 15 minutes away.

At 7.30pm, Ben called but she didn’t want to pick up. So I talked to her and told her that I’ll try my best to convince Nok to go to the dinner. I tried to talk to Nok and said that it’s Ben’s graduation that we’re celebrating so she should try to be nice and cheerful. Told her that she can get angry after the convocation. She decided to go but took her time shopping. We arrived at the restaurant late but thankfully, it was so full that the table that was reserved for us was still not cleared. We waited about 10 minutes before we could get our seat.

Oh boy, that sure was an awkward dinner we had. Nok was extremely quiet because she was still angry. Ben on the other hand, was trying to make peace with her sister and entertain her friend who flew to Ohio from Texas just to attend her graduation ceremony. But I finally managed to get Nok to participate in the conversation and she cooled off towards the end.

On Sunday, we didn’t get a chance to shop either because we passed by Legacy Village, which is a high class shopping boulevard. We saw Cheesecake Factory and California Pizza, which was very tempting. So we ended up spending most of the morning there, went for Ben’s graduation and came home. So much for planning…which is why I prefers spontaneity.

When we were alone on our back here, she said that I am very patient and does not have a short temper. What can I say? I grew up with a rascal that always wanted to win in every way. I could have complained about him but it would not work because he knows his way around my parents. I could have hit him back but it would not work either because he tends to be a bit dangerous at times. There were lots of times when I had to sacrifice days of studying or spending time on stuff I like to do just to make him happy. Everything was about him. So I learned that the best way to stay with him in peace is to suck it up. That’s how I became so patient and angry is a foreign word to me.

~satisfaction~

still stuck

I'm still stuck in the first step!!! There's two more weeks to ASM and I'm still struggling with the PCR. I definitely do not want to leave it hanging before going home to Penang. Will I make it in time for the wedding? I sure hope so because I don't want to leave the DNA naked at -20^oC. It’s going to deteriorate and by the time I come back, I might have to make new ones. That means all my work for one month would be gone just like that. I do need to complete the plasmid construct before I go back. Need more time!! Need to work harder!! *Red light flashing madly*

Now waiting for a PCR reaction with less amount of template and Taq polymerase. According to Kwi, I might have added too much polymerase since it also has 50% glycerol, which might affect the reaction. However, I am already using way less than 10% of the reaction volume. This is so frustrating. It’s not like I haven’t done PCR before. I’ve done it countless times and I’ve even troubleshoot it for different kinds of amplicons and templates when I was working with Sinorhizobium meliloti. Unfortunately, I have yet to work with such long amplicon and it seems like this is a lot harder to troubleshoot. Anyway, I am also running a positive control, in which I use a Verrucomicrobium genomic DNA as the template to amplify for the 16S rRNA gene. This should work since it works for everyone and I’ve used the same primers to amplify 16S rRNA gene of H. hepaticus when I was in the Young lab.

Gonna go eat my dinner for now and hopefully one hour later, I will get some products in the reaction tubes. Will have to wait till tomorrow to run the gel as the last bus is at 10pm and the gel is gonna take at least two hours.

ants

Ever since I had dinner, which is curry chicken again, I kept thinking about what late ah kong (my maternal grandfather) used to call me. He was the only one who called me that. No one calls me that anymore.

Somehow, the thoughts came to one sentence.

"U chi chiak ang hia ka chi liap bee"

(there is an ant who is biting a grain of rice)

It's part of a story ah kong used to tell while I was a little girl. Unfortunately, that's all I could remember and it keeps playing in my mind because I'm trying so hard to remember the story. It's so sad. I wish there's a way for me to freeze all the memories of me and ah kong spending time together. Maybe I can ask ah mah about the story.

~missing you~

stuck

Sad to say, the PCR did not work again. I have decreased my template to only 10 ng. All the annealing temperature I tried did not work either. Sigh..

I think I should try to cut the plasmid first and use the restricted plasmid as the template. Maybe it was just too complex to denature completely. Furthermore, my expected product is more than half the size of the plasmid.

oh well..will try again tomorrow with any other approach that I can think of. Time to go home to eat curry chicken!

~disappointed~

PCR

I'm back full time in Dr. Schmidt's lab. Last week was a short break for me since it was finals week and a few days of just looking through my previous results from the Schmidt's lab. Had a chance to go to Holland on Sunday with Nok, which I might blog about it later if I find the inspiration to. It was fun because we didn't plan it at all and I love spontaneity.

Ever since I came back to the lab on Monday, I've been doing nothing but running Polymerase Chain Reaction (PCR). I have to amplify a 4.5 kb long construct out from a 7 kb plasmid. I've tried a few times with different conditions and I haven't manage to see even a tiny bit of product yet.

1. Used regular Taq polymerase from Invitrogen.
2. Increased elongation time to 5 mins.
3. Used XL rTth DNA polymerase from Applied Biosystems.

Now running one with annealing temperature between 45oC to 64oC and I decreased the DNA template to only 10 ng. Hopefully at least one of these reactions will give me a product. Something for me to move on. This is only the first step in creating the broad host range construct and I'm already stuck. Really need to get this done before ASM since after ASM I would not have much time before I go home.

~frust~

coming to life

Flowers are blooming and the surroundings seem to come to life the moment the weather starts to warm up. It's no longer white and gray but full of colors. Cheerful colors from pure white to bright yellow to fiery red. That's the best thing about spring. The depression seems to just die away and be replaced by the need to be out there, enjoying the wind blowing against my face, to feel the petals of flowers dropping from the trees when the wind blows hard, to just listen to the songs of the birds and calling of the goose. There's a meaning to everything, unlike the cold, dull winter that sprung the loneliness often too many times. It's definitely a moment to be treasured. I couldn't stop taking pictures and everytime I press the shutter button, I wonder how can I capture the sounds of nature along with the beauty of it. Of course, it's impossible so I would just have to enjoy it right now. But I just can't help wishing that I could capture every detail of the moment for my parents to experience it.

~peaceful~

all done!

Finally handed in the final paper at 2.30pm today. Finished it around 10.30am but was not very satisfied with it. Unfortunately, I had too much of bile for the past few days that I can't seem to think anything else different from what I have written. So I took a break and resume editing at 1pm. E-mailed it to the three professors who will be grading it at 2pm and gave the hard copy at 2.30pm. Could I have done better? Possible but at this point, there's no point delaying it until the very last minute to hand in since I can't seem to make any major changes anymore. Nevertheless, I feel so relieved when I handed it in without having to rush.

Came home after that since I have no mood to start any experiments. The lab bench is still occupied by the previous student anyway. Since my desk was in such a chaos when I left it this morning, decided to give a good cleaning. Organized all the articles into EndNote and I realized that I have read 169 papers throughout the past 3 months. How did I do that? It's more than 1 paper a day and most of them were read critically enough to make comments and create hypothesis for follow-up experiments. I'm amazed at myself. It didn't feel like there was so much to read but now that it's all neatly piled up on my desk, it's definitely thicker than any text book I've ever read. But I'm glad I did because I've learned so much about microbial pathogenesis, from the mild Haemophilus ducreyi that causes chancroid to the deleterious Escherichia coli O157:H7, which is responsible for a deathly bloody diarrhea, and from oral pathogen Phosphyromonas gingivalis to gastrointestinal pathogen Salmonella enterica and even to sexually transmitted Neiserria gonorrhea. It is still overwhelming to be absorbing so much data within three months, especially on areas that I am not doing research on but it is better now than later.

As of now, I'm just enjoying the few precious hours of uninterrupted break. Time to start opening the covers of novels...

~relief~

soon..

It is a little less than 24 hours to the dateline of this final term paper - Utilizing Bile as an Environmental Signal and Bile Resistance Mechanism - is due. I am a little less than halfway through writing it and yet it seems so hard to start this next paragraph. I can imagine myself rejoicing tomorrow at this time, cooking up a healthy tasty meal that I haven't had in a while and enjoying a novel I've been itching to start along with the meal. But yet it feels like it is so faraway when this paper is still not completely written and edited. I've been reading about nothing but bile and its interaction with the bacteria for the past few days and why am I having such a hard time putting it all together in my own words? I just took a look at the last review paper I wrote, which was about 2 years ago - The Green Fluorescent Protein as a Reporter Gene - and I can't believe that I actually did so well in that considering that I'm still stuck with this paper. Will it be as good? I don't know but I sure need to finish it soon or else tomorrow will be a disastrous day.

~overwhelmed~