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.