音時雨 ～Regentropfen～

a postscript of my last post: that post was finished at Mar 13, 2008

Finally, I commenced my lab-work from this Monday. Now let me do a little summary on what happened in the three days.

Day 1. 2008/3/17

■Prepared all the needed solutions for DNA isolation.

■Frozen the three different plant leaves, Raphanus sativus (radish), Brassica napus (rape), and Arabidopsis thaliana (mouse-ear cress) which from another lab (the former two’s) and food market (radish’s), at -20℃.

■Isolated DNA from the three plant leaves in SDS method, (detail protocol was got from here: http://www.oznet.ksu.edu/wheatgenotyping/dna_isolation.html), and done to step 12. Detected by electrophoresis, (1% agar gel, 20ml, 1μl GreenView nucleic acid dye). Electrophoresis photo: 080317-5.

■[COMMENT] I just couldn’t be compatible with mercaptoethanol . For I failed on DNA isolation in last semester, I was nervous during each process. When I found some semi-transparent tiny filaments emerged after adding cold isopropanol for DNA precipitating, I got some hope. Later, after centrifuged my Eppendrof tubes, excitedly, I got a dot of white pellet. And when I done the electrophoresis, the bands of plant genome were seen. The place where A. thaliana’s genome should be is blank; I lost it, however. I also there were some small bands at the end of gel, were they degraded DNA or RNA (it’s could be, for I didn’t add Rnase)?

Day 2. 2008/3/18

■Firstly, I finished the left two steps of DNA isolation: centrifuging and getting supernatant.

■Prepare for PCR. Diluted my dry primers’ powder, by adding 100 times of water (account to primer’s quantity of “nmol”), and dissolving it at room temperature 20 minutes.

■Reaction system (10μl): Template DNA, 1μl; Primer 1/2, 0.5μl for each; 10×PCR buffer [Mg2+ free], 1; 2.5mM dNTP Mixture, 0.8μl; 25mM MgCl2, 0.6μl; Taq 0.4μl; add ddH2O to final volume of 10μl.

■PCR setting: [1]94℃ 5’ [2]94℃ 30’’ [3]52℃ 30’’ [4]72℃ 30’’ [5]goto “2,” 32 cycles [6]72℃ 4’ [7]4℃ for ever

■Electrophoresis order: λ-HindIII digest DNA marker D3403A, A, B1, B2, R, Ag, B1g, Rg (“A” for mouse-ear cress; “B” for rape; “A” for radish; “g” for genome DNA); photo: 080318 (failed).

■Therefore, another PCR system (20μl): Template DNA (diluted), 1μl; Primer 1/2, 0.5μl for each; 10×PCR buffer [Mg2+ free], 2; 2.5mM dNTP Mixture, 1.2μl; 25mM MgCl2, 0.9μl; Taq 0.1μl; add ddH2O to final volume of 20μl.

About diluting DNA: 0.5μl DNA(~300-fold) add into 10μl ddH2O, getting 1μl each time. And this time, I added another pair of been-verified primes to certificate my DNA template reliability.

■PCR setting: [1]94℃ 5’ [2]94℃ 30’’ [3]56℃ 30’’ [4]72℃ 30’’ [5]goto “2,” 39 cycles [6]72℃ 4’ [7]4℃ for ever.

■[COMMENT] All the electrophoresis photos showed faint bands, of the PCR products and DNA samples. The λ-HindIII marker was seen very strange. I knew afterward, that the λ-HindIII has got degraded for its long-time stored; the faint bands were since my little quantity of sample, also I used a unfit dye (I should have used bromophenol blue, but in fact, I used DNA loading buffer). Primers shouldn’t be wrong, so PCR’s failure may be caused by a invalid system (annealing temperature or something else), or one/more inactive constituents.

Day 3. 2008/3/19

■Electrophoresis order: Ditto λ-HindIII marker, AH, BH, BH, AK, BK, RK, Ag, B1g (“H” for another primers; “K” for my primers); photo: 080319-1 (failed).

■Eventually, I was recommended to use the Taq Master Mix, and also the two types of primer pairs. The new PCR system (20μl): Taq M.M. 10μl; Template DNA (diluted), 1μl; Primer 1/2, 0.5μl for each; add ddH2O to final volume of 20μl.

■PCR setting: [1]94℃ 5’ [2]94℃ 30’’ [3]56℃ 30’’ [4]72℃ 30’’ [5]goto “2,” 34 cycles [6]72℃ 4’ [7]4℃ for ever.

■Electrophoresis order: D2000 Cat #DM114 Lot #G5927 DNA marker, ah, bh, rh, ak, bk, rk; photo: 080319-2 (succeed).

■Preparing for next step—TA Cloning and transformation: test-tube, 30; plugs for test-tubes, 30; Eppendrof tube, 1 bottle; plate, 10; conical flask (150 & 250 ml), 2 for each; wrapping and waiting for sterilization.

■[COMMENT] Finally, PCR got succeed, (and I has waited so much times!). DNA marker I changed to D2000 Cat #DM114 Lot #G5927 (very clear but little inclined, which is caused by my gel-making?). Well, it will be my first time to do a TA cloning. Fight!

Day 6. 2008/03/24
For I got the wrong PCR products last weekend, so I edited the annealing temperature back to 54℃.

■In the afternoon, the electrophoresis told me that nothing was amplified out, strange!
■My tutor suggested me to build another reaction system, and to dilute template DNAs in another concentration(1μl DNA solution in 10μl ddH2O), also to use another primer which I mentioned ditto.
■PCR system (20μl):
Taq M.M. 10μl;
Template DNA (diluted 1/10), 1μl;
Primer 1/2, 0.5μl for each;
add ddH2O to final volume of 20μl.
■And a long PCR programme!
[1]94℃ 5’
[2]94℃ 30’’ [3]50℃ 30’’ [4]72℃ 30’’ [5]goto “2,” 2 cycles
[6]94℃ 30’’ [7]53℃ 30’’ [8]72℃ 30’’ [9]goto “6,” 2 cycles
[10]94℃ [11]56℃ 30’’ [12]72℃ 30’’ [13]goto “10,” 29cycles
[14]72℃ 10’
[15]4℃ for ever
■Electrophoresis order: B1, R1, M, B2, R2, B2+, (“1” for another primer, “2” for my primer, “M” is D2000, because I forgot whether I add DNA in tube “B2,” I made tube “B2+” by surplus mixture of PCR reagents); photo: 080324; 3.0μl sample + 1μl bromophenol blue.

■[COMMENT] Last week what I got from PCR were primer dimers, and that was why they were seen on the same level even though there were three DNA samples. Today morning’s PCR got failed again. I guess that was caused by unfit annealing temperature. The second PCR I did get some products, there were lots of primer dimers, however. The simple-like PCR is not so simple, sure enough.

Day 7. 2008/03/25

Ok, last PCR I got what I wanted. This morning I’ll do the ligation.

■The lightness of my PCR products looked almost like the D2000 marker but somewhat paler, so my tutor suggest me to use the ligation reaction system (5μl) below:
pMD-18 T-Vector, 0.5μl;
PCR product, 2μl;
ddH2O, 2.5μl
Solution I, 5μl
■Put the small EP tube at 16℃ for at least 30min.
■To get out a tube of 100μl competent cell, thaw on ice bath, then add 40μl, 40μl, and 20μl to my two ligation tube (rape and radish), and another positive control pUC 18 plasmid, respectively. Hatch the tubes on ice for at least 30min, to make sure ligation fragments have a good touch to the competent cells, (and I did ~45min).
■To put the tubes at 42℃ preheated water bath for 90s, (and I did ~80s), then put on ice at once for at least 2min.
■Add 0.9ml LB liquid medium, swing for 1h at 37℃, (and I did 1.5h).
■To centrifuge the tubes at 4000rpm/min for 3min, then I moved out 450μl of the supernatant, add 150μl of the rest to plate which mixed Amp.
■Hatch the plates at 37℃ overnight.

■[COMMENT] today’s ligation all seemed ok, but when I ended the centrifugation, I didn’t find any E. coli in the rape and radish tube, there is some at the bottom of the pUC-18 tube, however. Another embarrassed experience, I forgot to add X-gal and IPTG before I spreading the plates, I remembered it just after I done the spread! -__-|| Ok, well, besides the color screening, it doesn’t matter of that if I added them. Here, I only want to see something on my plate, no matter what it is!

Day 8. 2008/03/26
About little past 9, I checked my yesterday plates. Bad luck, only the radish plate had colonies. What did it was caused by? I don’t know. There were some probabilities, ligation failed, transformation failed, or Amp-resistance express failed, cells died, (for that, because in my lab, there are only copper-made spreaders, I’m afraid that if I don’t cool the spreader enough, the bacterium would be died by the high heat).

■Re-swing the tubes which concluded the bacterium for 1h 30min, at 28℃, (I couldn’t change to 37℃, for others’ stuff were also swing that time).
■Put out yesterday’s “empty” plates, open the UV light, sterilized for 1h, (I have the experiences of re-using “empty plates”, only because I don’t to waste, and there was nothing harmful to me [so far…]).
■I got 20μl from each tube, this time I remembered to add X-gal and IPTG.
■I divided three parts on a plates, spread my 20μl bacterium-liquid carefully into the three parts in order.
■Incubated the two plates at 37℃, then waiting for tomorrow. Hoping to see something.

Oh, today’s thirteen, happy birthday, YUUKA!

Yesterday afternoon, we did our graduation theses’ commencement report.
Of the lab which I applied in, in all, there were four professions and teachers who instruct us. For each tutor could instruct five students, the students’ number was much. Therefore, our report time was shrunk to about 5 minuets.
A little report re-shrink version of mine:

[TITLE] Construction of Expressing Vector for Antifungal Polypeptide
[MEANING] Antifungal peptide (AFP), is a small protein or peptide, which induced by fungal infection, and is produced by diverse organisms, include bacteria, fungi, plants, insects, and mammals. AFPs have potent activity to defend fungal infection. Its active mechanism is not the same to antibiotics, so that most fungi would not have restraint. To clone AFPs’ genes and combine them into expressing vectors, so we can product AFPs faster and better.
[RESEARCH SUBJECT] Isolate RNAs from Brassica nupus (rape) and some other plants; design a pairs of specific primers for RT-PCR; construct a expression vector (use PET30a plasmid); transform into E. coli and follow inspections.
[STEP LINE]
- Isolation of plant (rape & radish) RNAs
- Designing of specific primers
- RT-PCR to amplify AFPs’ cDNAs
- Electrophoresis and reclaiming purpose band
- Cloning to T-vector, sequencing
- Cleaving of T-vector and purpose expression vector (PET30a) by restriction endonucleases
- Electrophoresis and reclaiming
- T4 linkage of fragments and plasmids
- Transforming into E. coli
- Selecting recombine colonies and verifying by PCR
- Expressed proteins’ inspection
[REFERENCE] Antifungal Proteins, CLAUDE P. SELITRENNIKOFF, APPLIED AND ENVIRONMENTAL MICROBIOLOGY. | July 2001: 2883–2894.

The day before yesterday, I went out at noon. On campus, there were a lot of flowers had blossomed, and the sunshine was so warm that I got a very delighted.
In such cases, I always have some new floated ideals: “Ah! I must take photos of them, or they would be wasted of being-seen-in-that-time-only!”
Therefore, I, as a camera-girl, got been active last afternoon!
Since that time went into March, flowers blossomed one after another sometimes. Yet I noticed that the one sixth of a year had passed by, and how the elapse of time is really serious.
However, the best sign of spring must be flower, I backed with photos fully loaded. That night, I selected once and once again, eventually, I created a new album with the 17 good photos in.
Yet I embodied a somewhat long title, which I used a very cute font, in each photo; I also signed my beloved signature: “FairyAria feat. Kiyoshi.”
Chanchan~ I made them!

一昨日の真昼、ちょっと出掛けていました。キャンパスに、花がたくさん盛んで、お日さまがぽかぽかで照らしてくれて、すっごく心地よかったのです。
「ああ、ダメダメ！こんな景色を撮らなかったら無駄すぎる！」という発想が浮かんでた。
そして、昨日の午後、カメラガールの私が活躍しました！
弥生に入ってからというもの、いつの間にか花が続々と咲いていました。でも、「ああ、１年の六分の一がこんなにたったなんて」といきなり思い出して、「時間の経つはすっごく大変なもの」とも、気付いてなりませんでした。
でもね、春のサインには花限りというわけで、昨日の午後は写真満載でお帰りました。そして夜に、幾度といいかわるいかと考えて、最終17枚の写真が選び出しました。
そして、かわいい字体で一つやや長いタイトルを書いた後、ちょっぴりナルシシズムで、自分が気になる『FairyAria feat. Kiyoshi』でサインした。
ちゃんちゃん～出来上がりました！

Recommend to Hear—“dream scape”—by FictionJunction KAORI

Dreams use to be seen somewhat illusory, there is also likelihood of its coming true.
When we were children, we hardly ever stopped dreaming. If to ask a random person, “Do you have your childhood dreams?” the answers should not be only one, I think.
Me too, ever had lots of dreams.

—As far as I can remember, my first dream was to be a university student. Maybe now it no more sounds like a dream. For me who was in kindergarten that time, it was such a long span to when it comes true, that I even did not know if it could come true.
—I was attracted by beautiful clothing after I touched comic books. Therefore, I wanted to be a clothing designer. I can recall to that period clearly. Before our house moving, on one wall of my room, there my “designs” were be hung in line. I picked up my favourite clothing and draw them on paper, and scissored out by outlines, and glued them onto a paper tape. That was my Clothing Design Show. My handicraft got formed then, maybe, hum?
—In my 1, 2 grade in primary school, I saw a charming calendar at home. From then, I got a dream of becoming a photographer. However, in that time, we did not have any digital camera, so my parents not used to ask me to take photos. You can soon find “my works” in our former albums, by recognizing the incomplete people.
—I also dreamed to become a scientist vaguely, even though I did not know much in science. Because I was only a primary student, all the science lessons, such as biology, physics, and chemistry, were all include in one lesson which named Science. Which field did I prefer to, now I have had no idea. There is one thing almost certainly, whatever I chose, nature scientist was must included.
—In my middle school days, I admired to be a radio announcer, (especially in a classic music channel). I did not get the fact until high school, that to become a announcer is extremely very difficult. As a result, I gave this dream up.
—I got strong passion on geography in high school grade one. However, biology gained my more passion, so that, after many dreams, I chose science.
Finally, one dream got come true.

I still had some other tiny dreams, some of them have began to be forgotten. Like for example, to be a teacher or a linguist, a cartoonist or a chorus member, a world-round guide or a novelist, etc… I never wrote them in my diaries, but only need to recall a bit, like a blast, they all start to turn in my sight, round and round.
I cannot forget them yet they are only dreams.
Anyone must have lots of dreams in childhood. This and that, all the occupations, also fantastic works, all of us do, ever dreamed some, didn’t we? Time goes by, we began to forget some, also we got enough knowledge of the impossible, and we only meet them in our reminiscence; we maybe lose passion on ex-dreams, or maybe select other way to go, so the ex-dreams got sleeping into a corner of our memories. Anyhow, we all had the period, the time when we had the most passion on life, the time when we used to dream something, the time which can gets our heart full of memories. Now, to ask yourself, even if it maybe tiny, do you still have any childhood speck in your mind?

夢は儚いものだと言われるが、夢を叶える可能性があります。
子供の頃、ほぼ全ての子供も同じく夢見がちだったのです。「あなたの子供だった頃に、どんな夢がありましたか」と、随意に誰かに聞けば、返事はたった一つではないはずです。
私も、こんなそんな童夢を持っていました。

――憶えているものの限り、私は一番なりたかったのは大学生です。これは今に聞き返すとぜんぜん夢なんてだと思えないかもしれませんが、幼稚園時代の私にとって、まだ遥かすぎて本当に叶えるのかさえも未知だったことです。
――漫画などとさわった後で、きれいな服装に誘い込まれて、服装デザイナーになりたかったことです。これはちゃんと憶えています、引越し前私の家に、部屋の壁に掛け並べている「私のデザイン」がありました。私は紙で漫画に気になる服装を書いて、そしてそれをはさみ出して、一本の紙帯に張り付いて「ショー」しました。多分、私のすごい手仕事能力はその頃から育ちだしましたね。
――約小学校１、２年生、家にあるカレンダーから、素敵な風景写真を見ていました。あれから、夢にまた、写真師になりたかったです。でも、あの時今のようなデジカメラがなかったので、家族はほとんど私に写真を撮らせなかったです。昔のアルバムに、私の作品ならすぐ見分けます、不完全な人がいるですものね。
――科学にまだあんまり知らなかったくせに、私はただ科学者のようなぼんやりとした夢がありました。せいぜい小学生だったから、生物や物理や化学や全部理科に囲まれていました。いったいその時、私は何の科学者になりたかったのか。たぶん、九分九厘、自然科学者かもしれませんでした。
――中学時代、ずっとラジオ（特にクラシック音楽番組の）アナウンサーを憧れていました。でも、高校生になった後で知りました、これは難しくてならないことで夢のままに置かざるを得ませんでした。
――高校生になった私は地理に興味が湧いたことがあります。でも、生物学はより私を情熱を燃えさせていました。結局、いろんな夢見た後で、私は理科を選びました。
ついに、一つの夢は現になりました。

それ以外、ちいさな夢や忘れてしまった夢もまだたくさんあります。たとえば、教師とか言語学者とか、漫画家とかコーラス部の部員とか、世界観光のガイドや小説家など。こんな夢は私も日記に書いていないけれども、少しだけ思えばみんなきゅうに目の前でぐるぐると廻り続き始めました。
夢だけど、忘れられないんですね。
誰だって、子供の頃にたくさんの夢があったのでしょう。あれこれ、あらゆる仕事、また空想ほどの仕事、いくつかを夢見たことがあるのでしょう。時間の経つとともに、一部の夢が忘れかけていたり、一部の夢は不可能だと知ってその時代の想い出になったり、一部の夢は興味を失っていて或いは進路に選ばれなくて記憶の片隅に置いていたり、と言うことになりました。みんなそれぞれその時代を持ったことがあります、その一番生活に無垢に熱心していた時代、その夢見がちな儚く遥かな時代、その誰にも胸がいっぱいに満ちれる時代。自分に問いましょう、いは自分の生活に、子供時代の片影さえでも、まだありますか。

Heroin, the loanword which from English caught my attention recently.
Why was the word constructed by “hero” and “in.” Doesn’t it seem strange?
To say why, for that hero means a brave man, and the suffix “-in” means a kind of proteins. Don’t you think… it’s such a kidding when they are putted together. What, a kind of heroes’ protein?
In spite of that, when I checked up once again I got everything. The word derivation of heroin comes out from a German trademark. Oh, look, it’s not from English, what a pity!
Well, apparently it not means “heroes’ protein.”
Aha.
Sure enough, there is something importance and interesting of investigation of word derivation.

ヘロイン、英語からの借用語で、本当の姿は「heroin」、この単語は最近私の気を取った。
「hero」と「in」、どうしてこんなに構築なのか、こんなで変なのよ。
だって、「hero」は英雄の意味で、「-in」は接尾語の一つ、「○○蛋白」などの意味がある。この二つを一緒にすれば可笑しいんじゃない？「英雄蛋白」じゃない？
でも、今もう一度調べてみると全てが分かる。この単語の語源は、残念ながら、ドイツ語で英語じゃない。これはある登録商標から来た単語である。
そうなんだ、やはり「英雄蛋白」じゃないね。
あはは。
語源の調べは面白くて大切なのね。

I’d finished my fifth picture album four days ago, its name is Sherbet. In my LINGOES dictionary, there are two meanings of Sherbet, and I prefer the America one, of that sherbet is like ice cream but made with fruit juice, sugar, and water, (the British meaning is that a sweet dry powder that tastes fizzy and is eaten as a sweet). Well, it sounds aromatic and delicious, doesn’t it? Say about why I chosen the word for the album, I met it just by accident, when I was browsing a dictionary, (I like it, because I can always meet some interesting words). However, there is nothing deeper between the single word and my pictures—it just a name. You see, anything of we have does need a name, or we would think it lacks some exist-feeling. Any word your mind catch up could be a name, and you named something by the word(s), then this stuff becomes perfect to you. There is such the consciousness of that you do know it belongs to you. No matter of that how absurd the name is: Kipper or Bench for a cat, Police or Genesis for a band, or Opera for a browser (^o^). Naming is a wonderful program, after it some unrelated conceptions got linked, so the namers and (or) the owners accept them into their lives. Everything got perfect, harmonic.

Hey, what did I say here? Ok, just think it an useless trivia narration.