音時雨 ～Regentropfen～

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.

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.

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.