音時雨 ~Regentropfen~

♡ Little Light, Eternal Light (JP)

最近1番お気になるアルバムは、KOKIAさんの「The VOICE」です。
KOKIAさんの歌は、今まで多く聞いていなくて、ここでなんだかあんまり、うまく言えないような気がする。でも、私の聞いた歌の範囲では、このアルバムがどうしても前列に立てられる。
「The VOICE」は、KOKIAさんの十周年の記念アルバムとも言える。ファンたちへの、真心からの優しくて暖かい贈り物。全ての歌も素晴らしい。KOKIAの変わり多くの声もこのアルバムでよく感じられる。
キーワード:コエ、ウタ、オモイ、アイ
[01]穏やかな静けさ~浄歌
ジャケットの雰囲気が1番目の曲とぴったり溶け込める。深い青さに、KOKIAの繊細な声が少しずつ耳に沁みこんで、次から次へと、私たちを無限なる悠久に包み込んでいる。
――穏やかな静けさが心に宿りますように。
[02]Follow the Nightingale
あるゲームのテーマソング。これと関わらずに、とても立派な、いいえ、かっこい歌だと思う。「調和」と似て、雄大なエピックそのもので、神秘満点の自造語(?)で歌い出された豊かな旋律。
――EKUDUS TINOTA IAM
[03]Ave Maria
暗い暗い聖堂へと降り注ぐ初めての朝の光のように、高い天窓から天使たちが柔らかい愛と安らぎをくれました。アベマリア、神様が吾ら至福をくれますように、いついつまでも。
――アベマリア
[04]届きますように
無くなってしまった友達への思いが、届けるかどうか不確かなものだが、もし本当に届けたのなら、光に包まれるたび、風に吹かれるたび、あなたの心を感じられた気がする、きっと。
――From your friend.
[05]song of pocchong~雫の唄
奥山に精霊たちの泉がある。透明で涼しい雫がいつも軽く石に落ちていて、微かな唄になる。速くて緩くて、高くて低くて、深くて浅くて、至純な自然を求める人しか聞かない、精霊たちができた魔法の唄。
――lek wistru leiam lek wistru lekiam
[06]ごめんね。
誰かも過ちを犯すことがある、不意に誰かを傷つくこともある。何もかも言葉で伝えきれないけど、この謝る気持ちが言葉にしないと、伝えられなくて…
――肝心な時に意地はって言えなかった たったの4文字の優しさ
[07]Lacrima
聖なる涙が乾くなるとき、人はより強くなれると信じる。オレンジの夕日が沈んで、瞳から反射するのは、止まらなくて満ち溢れる想い。そして新しい笑顔が次の太陽に照らされるのが、強さがあるから。
――弱虫なんて誰も思いやしない あなたは既に充分がんばってるから
[08]何もかもが星になって
世界中に無数の人たちが自分なりの世界を完璧になるため、ほかの人と戦うことを逃せない。愛と正義などは元々不確か過ぎるもの。誰もいつか星になるから、どうかその前、平和な世界を作れますように。
――虚しさだけが空を斬るのはいたたまれないから 何もかもが星になる頃忘れ去られてしまうの?
[09]il mare dei suoni (音の海)
音楽は不思議なもの、心の純粋な人しか作れないもの、天からもらった永遠になれるもの。音楽を好きになれることが不思議。音楽が自身の広さを全てを包み込まれる。音楽の海に生まれられて、幸いです。
――un suono caduto nel mondo in blu. un dono dal cielo. (青い世界に落ちた音 空からの贈り物)
[10]everlasting
あなたは永遠を信じますか。二人が心を分かち合えたら、愛が二人の間に生まれるから。生活が明るい愛の光に包まれたら、私たちが生き続ける。世界が前へ行けたら、いつか永遠に触れますから。
――I wish this world to be freed of sad and wrapped with eternal light.
[11]小さなうた
飾らぬ人々の生き様が歌のように、小さいだけど強い。歌に込まれる想いが日々の流れと共に溢れて、ついに、小さな旋律が「一つ」になれて、この世界をより優しくなってゆく。
――every day every time 歌い続けたい 溢れる想い止まらない
[12]「私にできること」
私たちがこの世に落ちる以上、きっとできることがある。これがどんなに小さなことだって、きっとこれを受け取る人がいる。だから、できるだけ、潔く、生きよう。
――届いてほしいこの気持ち あなたに贈る歌
ちょっとだけですが、もし「say goodbye & good day」を最終曲にすれば、より完璧だと、私はこう思うの。あなたはそう思わない?

❀ Deepening of Strains

Ok, from now on, I will not write more about what I did, but what I thought.
Now, I finally realized how important different strains are. Recently, I extracted plasmids once and once again, for later digestion and ligation. But I couldn’t extract them well even in case of kit using. Then by accident, I found that some E. coli strains (like JM and BL21) need to dispose the too much proteins especially the DNase, for such strains may product more which may lead to a result of plasmid DNA degradation; other strains (like DH5α) need not.
I remembered when my tutor gave me the vector plasmids, he made me to transfer the pure plasmid into competent E. coli, and then he added: “you may notice the strain, even though the competent cell are all E. coli, the different strains have different features.”
Then I only knew that DH5α is more efficient to express toxin proteins, but now I deepened my understanding.
Last time I transferred the plasmids into strain BL21, and later I always failed on plasmid extraction. Now I know why—BL21 expressed more DNase so that my plasmids got protein contaminated and degraded itself.
At lab, I have learned more delicate things which are not printed on books.

♡ Real-time Lab Trace [part. 7: Apr. 14-19]

Day 25. 2008/04/14
Today’s all results were strange. The plasmids extracted by Kit not so pure, because there were two bands stay at near positions where my plasmid should be. Although I took about four hours to digest, of the five samples I only got one MAY BE what I want.
So that tomorrow I will have to extract again, by hand and Kit respectively.
Day 26. 2008/04/15
I extracted the plasmids by Kit, but the photo was still not very perfect (080415-PLASMID.jpg). Maybe my gel had something incorrect.
XhoI digestion was not good too. When I checked my primers in the evening, I got very surprised. My sense primer not concludes XhoI site, but EcoRI and NcoI!
Now, the problem got solved.
Day 27. 2008/04/16
Digestion 1 (I got the enzymes a wrong quality, which should be 0.5, but I added 1, so the concentration was wrong):
B1 & R1: XhoI 1; BamHI 1; 10×K Buffer 1; ddH2O 3; plasmid 5;
B2 & R2: XhoI 1; NcoI 1; 10×K Buffer 1; 10×BSA 1; ddH2O 2; plasmid 5;
R3 (only a little test for another two sites on pMD-18): EcoRI 0.5; HindIII 0.5; 10×M Buffer 1; ddH2O 3; plasmid 5;
But the cut bands were so faint that I couldn’t see it well without Photoshop editing (080416-digestion1.jpg). I guessed it was because of that I added more plasmids.
So…
Digestion 2
B1 & R1: XhoI 0.5; BamHI 0.5; 10×K Buffer 1; ddH2O 6; plasmid 2;
B2 & R2: XhoI 0.5; NcoI 0.5; 10×K Buffer 1; 10×BSA 1; ddH2O 5; plasmid 2;
R3 (only a little test for another two sites on pMD-18): EcoRI 0.5; HindIII 0.5; 10×M Buffer 1; dd H2O 5; plasmid 2;
This time the bands were little lighter (I only detected two of them), but still faint. So I left the other tubes to digest overnight.
Waiting for tomorrow’s electrophoresis.
Day 28. 2008/04/17
The overnight digestion wasn’t more ideal than 1-2 hours reaction. I even couldn’t find any bands on the gel!
Sure, that’s only a test, what I should to do was to extract enough plasmids, and this time I also used the Kit.
But…
Oh! My goodness! I forgot to change the spin column onto another EP tube, so I had to precipitate plasmid DNA by ethanol that afternoon. Good luck, the result was not bad.
Then, from my tutor I got 10μl of E. coli which including the expression plasmid pET30a, and also 1.5μl of pure plasmid pET30a and pGEX6-1. I transferred the 10μl bacteria into liquid LB with Kan.
Day 29. 2008/04/18
I did a 100μl digestion for later gel extraction, (080418-digestion2-100ul.jpg).
The pET30a bacteria didn’t grow! I added some LB into the original EP tube, swung for 1 hour, then transferred into antibiotic free LB.
Day 30. 2008/04/19
The antibiotic free LB got some bacteria, but LB-Kan didn’t turn cloudy, so I had to transfer the pure plasmids into E. coli.
Maybe later I will try to make competent cells, so I needed to preserve some plasmid free cells. I added 35μl competent cell into the pGEX6-1 and pET30a vectors respectively, and the last 30μl of cell I transferred into antibiotic free liquid LB.

❅ Real-time Lab Trace [part. 6: Apr. 9-13]

Day 20. 2008/04/09
Now, I got an maxim, that my bacteria will not grow enough until at least two nights. However, of the test tubes I re-swung yesterday, still only A6 and B1 turned cloudy. From the old plates the single colonies all grew to cloudy. So I did a colony PCR by some of them.
Ok, here, I will not mention anything about what I said above. I got 3 single colonies from Radish and 2 from Rape.

Day 21. 2008/04/10
In the morning, I saved the screened out colonies by added half volume of glycerine. Then I extracted the plasmid again, but still I didn’t got enough.
Maybe because that I didn’t dissolved the DNA pellet completely. I’ve decided that I will re-deposit the DNA tomorrow.

Day 22. 2008/04/11
Hey! I knew why I always got faint bands of plasmids! It was because that I didn’t dry the DNA pellet enough. In other words, I added ddH2O into the tubes when ethanol still stayed with DNA. Nevertheless, DNA which mixed with ethanol would lead to diffusion while electrophoresis detection—that’s why the bands were always faint.
That fact I have testified in the morning, and also in the afternoon. I saw the lightness of bands was like the maker. However, recently, I also found that the marker lightness was very faint. Another classmate in the lab also got the same result. So I think the faint band was caused by marker itself.
Tomorrow I will try to extract plasmids again, and with Rnase. Then I will try to digest the plasmid by XhoI, to testify my target fragments do have been transferred into the T-vectors.

Little PS: when I did the today’s second electrophoresis of plasmid, our GreenView, the nucleic acid dying, was used up! The new tube would not be send until next Monday. So one of the seniors told me to add some TAE buffer for washing. So I added 20μl to wash the tube wall and the inside of the tube cap. Then I added more dying into my gel liquid. Thank goddess, I could see my bands!

Day 23. 2008/04/12
I extracted the plasmids from two rape straits and three radish straits in the morning. In the afternoon, I expanding inoculated two radish strains, R1 and R2, but then our swing bed was used by others at 30℃. I had to incubated at this temperature. I also used Xho I to digest two of the plasmids for about 3 hours.
Now, the electrophoresis photo showed me that, I failed. Also the Xho I digested tubes.

Day 24. 2008/04/13
I couldn’t sure if I digested successfully, so I used one tube to run PCR; as the positive control, I used the plasmid liquid, and the negative control I used another plasmid. Then I expand inoculated the five straits again.
Sure enough, the 30℃ incubated bacteria didn’t very grow robustly. However, I extracted plasmids from all the five tubes of E.coli: B1 and 2, R1-3. For each time I found the RNA fragments stayed at the bottom of the gel, although I added RNase, so I used different ways to extract this time.
By Plasmid Extracting Kit: B1, R1;
By hand: B2, R2;
By only use the Solution with RNase of the Kit, and the other solution were made by me: R3.
After extracting, I got 8μl to digest, with 1μl buffer (10×) and 0.5μl Xho I and 0.5μl H2O. It last about one hour.
Ok, all’s ready, I would detect them by electrophoresis!
The result showed: the two plasmids extracted by Kit were good and no any RNA remain. These by hand were also extracted out but there were RNAs, although I added RNase. About PCR products, the digest product one got the target fragment, and another unspecific fragment, which I guessed it’s because of Xho I digesting; the negative control didn’t amplified out any fragment. The Xho I didn’t digest out any! Maybe the reaction time was too short, I think, so the first thing in tomorrow morning is, digest my plasmids for enough time! I want to use a whole morning about 4 hours to do it.

✾ Real-time Lab Trace [part. 5: Apr. 4-8]

Day 15. 2008/04/04
What?! This noon I found some colonies on my plates, of which both the new X-gal spread and the more old X-gal spread, showed blue color! So that it must be that the former X-gal had lost its activity, and finally, I saw the beautiful and lovely blue of the colonies!
The Amp-free plates got a lot of colonies, contrary, there were almost not colonies on the Amp plates. There were nothing on the empty transferred plate; the pUC-18 plate got the full plate of tiny colonies. They all verified that my transformation was succeed; however, there are a few colonies on the radish and rape ligation plate, showed that most of the bacteria died on the Amp plates. Was there anything wrong in my last ligation? If it were, I would say that I got a deficient ligation time, which at least one hour but I only maintained 45 minuets at most… but even though it may be wrong in the ligation time, the un-ligated empty T-vectors should be transferred into cells, then show lots of colonies on the plates, just like the last time… well, the more I thought, the more I got puzzled.
At last, I make four new plate, with two Amp-free and two Amp added, each of them was covered by X-gal and IPTG (40:16). I spread all my remained bacteria liquid, including the liquid of this and the last time, onto the two Amp-free plates after 1.5 hours’ swing. On each Amp-added plates, I transferred 8×5 colonies from my last plates, one radish plate and one rape plate. I wanted to find some recons from the last plates. Then I transferred four single colonies from the new grown colonies, one blue from radish, two white from radish, and one white from rape, into four test tubes, then I swung them overnight for tomorrow’s PCR.
Ok, hoping to see recons, I also want to try endonuclease digesting earlier!

Day 16. 2008/04/05
This morning, I got out my plates. Now I got some new colonies which came from my second transformation. They are almost white, but it can’t instead of a succeed transformation, for some may be fake positive.
I did a colony PCR, used the bacteria liquid I swung yesterday.
I collected some colonies from the two Amp-free plates, transferred the colonies into 8 test tubes, radish tubes and 4 rape tubes. Then the PCR result showed that two of the four samples contained my target fragment, (electrophoresis photo: 080405-ColonyPCR.jpg). So that I see a beam of light toward the next step. Once again, I transferred another 12 (or 13?) single colonies into test tubes.
Ok, tomorrow, another batch of colony PCR!
Here I have a strange problem, which about plasmid extraction reagent. Solution I is a mixture of glucose, EDTA, and Tris-HCl, all the manual say that it should be autoclave sterilized. Glucose is unstable in high temperature, so its sterilization should be controlled. According to Molecular Cloning III, I set the autoclave 115℃ for 30 minutes, but I found the solution turned to brown-yellow after sterilization. Why~~~

Day 17. 2008/04/06
Today, I did only a colony PCR, used the 15 samples I swung yesterday. Nevertheless, no any amplified fragments…
In the afternoon, I spread the 8 bacteria mixture. When I centrifuged them, there was only a tiny white pellet on each bottom of Eppendorf tubes. It like that I did failed on last ligation, most of the bacteria was killed by Amp. However, I will screen the Amp-resistant colonies, so I had to spread the little liquid, with X-gal(70) and IPTG(16).

Day 18. 2008/04/07
All my yesterday re-collected bacteria got growing failed! Did the Amp was too toxic? I left the plates in the incubator so that they, which still alive but just not grew into colonies (if there were), could grow freely.
I was afraid that I failed to detect the bands, because I didn’t do the electrophoresis well, I re-PCR some samples of them. Nothing, however…
Then I got 2/3 of the target-contained two test-tubes of bacteria into 4 new tubes. I want to expand them. I also streaked the two strains on a plate—hoping to get some single colonies.
In the afternoon, I transferred single colonies once again, from the three plates:16 of rape II, 3 of radish I, and 4 of radish II. Later, I spread a few liquid. I will do colony PCR tomorrow, also the expanded two.

Day 19. 2008/04/08
For that I have got the recons of radish-AFP, now I need to screen some from rape. Nevertheless, I did a colony PCR, used the DNA templates from tube A3, A5, B3, B5, C3, which were samples from rape; at the same time, I also tried one of the samples I expanding inoculated yesterday, for testify whether I expanded the straits successfully.
While I was doing colony PCR, I tried to extracted plasmids of the expanded two straits.
At noon, the electrophoresis photo told me that I didn’t got my expected fragments beside the expanded one, and once again, I found only faint bands of plasmid…
Was it caused by the not-enough bacteria? The liquid LB medium in each tube all showed a clear yellow, just looked like the un-inoculated LB color. So that I had to swing the test tubes at 37℃ again. This time I only swung the rape tubes—I must get recons of rape! About 13:00, I swung them, and at 15:36, I found two of them turned cloudy, which showed there were bacteria grew! Ok, I got a little relief. After streaking the two bacteria liquid, I left lab complacently. The next is relied on my colony PCR of them!

♢ Real-time Lab Trace [part. 4: Mar. 27-Apr. 3]

Day 9. 2008/03/27
One of the two plates got all the three bacteria colonies, (and some of the colonies grew together, became to moss… oh, my, this time they grew too robust!)
■Chose some single colonies and transferred to liquid LB medium test-tubes, incubated at 37℃ by swing.

In the afternoon, I tried colony-PCR first time. I originally only wanted to test 15 samples (colonies), but, what’s wrong with me, I made a PCR mixture which enough to do 25 samples, (I think that I must have got jumbled)
So that I had to do 25 samples; the tiny tube tips made my finger pain! After putting the tubes into PCR apparatus, I clocked for 1.5 hours for prepare the gel. Everything seemed OK this time, but when I saw it about 1 hour later… my PCR had been stopped… who did it!? However, only God knows…

Day 10. 2008/03/28
In the morning I tried a raw-test for the incubated bacterium in test tubes.
However, I saw nothing but RNA fragments… so that I wanted to extract the plasmids in the afternoon.
I got some solution I, II, III from a classmate who also extracted plasmids then. This time when I added about 1ml bacteria liquid, I also added 0.5μl to each PCR reaction tube. This afternoon I passed in a full and busy tempo—extraction plasmids, amplifying my target fragment DNAs by PCR, before the end of PCR, I also made a gel for the later electrophoresis.
This afternoon I got nothing but RNA fragments, however…
I don’t know which step I got missed, the plasmids should be extracted well in theory…well, try again in the next week!

Day 11. 2008/03/31
■Add some fresh medium to each test tube to let them grow more, swing at 37℃ for some hours.
■Did another PCR by another four single colonies.
■Extracted the plasmids once again.
But still I got nothing but RNAs, neither my extracted plasmids or the PCR results…

Day 12. 2008/04/01
Why I always get nothing from my plasmid extraction? No matter of what the plasmid they are, at least I should see the empty vectors which survived on the Amp added plates. Well, this morning, I did it again, and this time, I subtracted the last step of ethanol washing. Then I detected by electrophoresis, I get some bands this time, however, they are faint, very faint… and me too… (see the mixed photo: 080401-plasmid_ps.jpg)
Another painful experience, of the blue-white screening, I got nothing showed blue, it’s impossible that I could get all the colonies absorbed the ligated vectors and then grew to recons. I doubted that it might be caused by I added not enough X-gal and IPTG… so that I made a mixture of X-gal(80μl) and IPTG(32μl), then spread half to two plates. After the liquid dried, I picked some single colonies, and transferred each to the two plates. I almost repeat the cycle 100 times: burn for sterilizing, cool it and pick out a single colony, transfer to another plate… not difficult but trouble…
Ok, I incubated the plates at 37℃, oh, colors, I want to see you!

Day 13. 2008/04/02
Maybe the X-gal got something wrong, yesterday’s plates still didn’t show any color.
My lab bought another type of competent cell, DH5α. This afternoon I used it to do another transformation. The reaction was the same like last time.
After transformation and one-hour swing, I added 65μl X-gal and 8μl IPTG into each tube. For I only had two plates then, I had to spread the four tubes of bacterium, (transformation of Radish, Rape, pUC18, and a empty competent cell), into the two plates, and each bacteria I got 85μl for spreading, (hoping it was ok -__-||).

Day 14. 2008/04/03
Hah! There was nothing on the plates when I came to lab this morning. Ok, E. coli, I’ll give you some fresh medium LB and swing you and spread the plates once again!
■This morning I prepared 5 new plates.
■At noon, I mixed X-gal and IPTG in proportion, (40μl:16μl), and this time, I changed to another new tube of X-gal. Then I spread the mixture of 56μl on the surface of my LB plates.
■Waiting for a hour, I got the re-swung tubes, centrifuged, discarded the supernatant and remained about 200μl of it; spread each plate by adding 120μl of bacteria liquid.
■When I put the plates into the incubator, surprisingly, the pUC-18 transferred part got a large of tiny colonies! But the other parts didn’t get, why?
■PS: I made four Amp-contained plates and one Amp-free plate, for to verify the “empty” transferred bacteria is still alive. I also spread the bacterium which I saved last time, (about 20μl in each tube).

■[COMMENT] I felt that during the two weeks, I still repeat the same work: ligation, transferring, and screening. I even didn’t budge little forward. It was not good, however: the extracted plasmids was too few to continue, the transferred cells grow not well, the blue-white screening was no any effect, and the worst is that I don’t know what’s the root of them. Expecting to get some steps upper …

☆ Real-time Lab Trace [part. 2: Mar. 20-21]

Day 4. 2008/3/20
■TA Cloning step 1: Ligation (in the morning);
■TA Cloning step 2: Transformation (in the afternoon);
■TA Cloning step 3: Hatch the transformed cells (overnight).

■[COMMENT] It seemed that my experiment got messed hence ligation. Firstly, I used more pMD-18-T vectors than what I should have to use, as well as my PCR products, (therefore, I used all my PCR product which from radish). Secondly, I forgot to make my ligated products and the competent cells enough touched. So that this TA cloning must get failed.
Day 5. 2008/03/21
■Because of the failure of yesterday, I had to amplify my target fragment DNA by PCR one more time. At first I wanted to try Touchdown PCR, (in the morning; by changing of Mg2+, annealing temperature 54℃, but all the tubes got nothing (why?)!
■For the first PCR, which I ran in the morning, got failed, I guessed that there may be some problems with the Taq M.M. There are two tubes of Taq M.M. in the freezer, (their code were Lot #G5910 and #G5409), and in my succeed PCR, what I used was the #G5409 one. However, today I used the other, (I didn’t know there were two tubes until my failure of morning PCR). So that I tried another PCR, and I used all the two Taq M.Ms, diluted some fresh DNA (although it may be not necessary).
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: B1, R, B2, D2000 marker, A, B2; photo: 080321; 3.5μl sample + 1μl bromophenol blue (somewhat strange).
■[COMMENT] Really, there were my amplified products, and each DNA template got its product. Nevertheless, this time I found that the position of bands got changed. They looked very, very strange and questionable. In a word, these products were not the same as last succeed one. I got only smaller bands, and all the template got the same band! Why?! I should have gotten the correct bands like last! Here, wait a moment! In this PCR, I set the annealing temperature of 56℃. Did the higher temperature made the amplified fragments incorrectly? Well, I will verify it on next Monday by another PCR which be set a lower annealing temperature.