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