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.
■[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!