Team:NTU-Singapore/Notebook/15 July 2008



  

=Tuesday 15 July= {|border="1" style="background-color:#ffffcc;" cellpadding="20"
 * All 4 plates of Fe-GFP from Monday had colonies. We did the inoculation in the afternoon.
 * Ligation of T7ptag, E7 with empty vector from GFP:
 * We already got E7 (cut with E/P), E7 (cut with X/P) and T7ptag (cut with E/P) from Monday.
 * 1140: digestion of GFP plasmid with EcoRI/PstI in Buffer 2;digestion of GFP plasmid with XbaI/PstI in Buffer 3; supD with EcoRI/PstI in buffer 2. Incubate for 1 hour from 1225-1325.
 * 1325: MinElute PCR purification for the above digested plasmids.
 * 1355: gel run for the 3 digested plasmids from Monday and 2 plasmids digested at 1140 (supD was not ran because a 2% gel must be used instead). Results: T7ptag,E7(X/P) and 2 GFP vectors were gel extracted. However, the bands were rather smeary. Later gel extraction followed by nanodrops showed very low concentrations. Therefore, we decided not to ligate today.
 * We also tried cutting E7 with E/P in buffer2 for 1 hour (as the one we used for ligation today was cut on Monday using EcoRI buffer) After digestion, QIA PCR purification and gel run, we found that the 2kb bands of E7 were good enough to extract out of the gel. We'd do that on Wednesday.
 * 1725-1930: transform of the succesfully ligated LacI-RBS34 and LacI-GFP plasmids into top10 cells.
 * Inoculation of Fe-GFP (3 colonies each plate x 4 plates); GFP (6 colonies).
 * Inoculation of Fe-GFP (3 colonies each plate x 4 plates); GFP (6 colonies).