Team:NTU-Singapore/Notebook/10 July 2008



  

=Thursday 10 July= {|border="1" style="background-color:#ffffcc;" cellpadding="20"
 * 1015-1215: Hung, Lu Chao: minipreps and nanodrops for LacI-GFP 1ul (x3), LacI-GFP 4ul (x2), LacI-RBS 1:3, LacI-RBS 1:6, Fe-GFP 1ul(x3), Fe-GFP 4ul (x1). The microliter amounts specify the amounts of ligation mixtures transformed into the cells.
 * 1015-1215: Hung, Lu Chao: minipreps and nanodrops for LacI-GFP 1ul (x3), LacI-GFP 4ul (x2), LacI-RBS 1:3, LacI-RBS 1:6, Fe-GFP 1ul(x3), Fe-GFP 4ul (x1). The microliter amounts specify the amounts of ligation mixtures transformed into the cells.


 * 1230-1300: Hung: prepare digestion mixtures (cut with EcoRI and PstI for gel check) of the above plasmids. Incubate for 2.5 hours.
 * 1400-1415: digestion of E7 X-PH (XbaI-PstI His tag) and E7 E-PH. Both plasmids were gel extracted on 9 July and purified on 10 July. DNA used for each mixture was 28 ul, and total digestion mixture was 40 ul. Incubate for 3.5 hours.
 * 1530-1600: Min: run gel check for the plasmids digested at 1230. Later gel results showed that LacI-RBS 1:3 is in fact correct. However, all the LacI-GFP and Fe-GFP plasmids were not the ligated plasmids. They were just the uncut vector (no 1kb and 3kb bands were seen, only the 2kb and around 250-300bp bands). This again arose the questions about ligation efficiency. As for LacI-RBS, although successfully ligated, the odd of getting ligated plasmid cell was 1 in 12. Most of the cells only contained the uncut vector.
 * 1530: Darius: gel extraction for T7ptag and supD which were digested on 9 July.
 * 1745: Hung:PCR purification for E7 X-PH and E7 E-PH (digested at 1415).
 * 1800: We intended to do ligation, then transformation. However, it was suspected that the LBA plates prepared yesterday, and also today were faulty (as top10 cells seemed to grow on these plates). Therefore, we decided to take our time to draw the detailed plan for Friday experiments, when we will prepare all the inserts and vectors required and ligate on the same day. Meanwhile, Min & Lu Chao ran a mass PCR of E7, T7ptag and SupD...