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.
■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).
■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.