Team:NTU-Singapore/Notebook/21 July 2008



  

=Monday 21 July= {|border="1" style="background-color:#ffffcc;" cellpadding="20" Hung, Lu Chao:

Ligation of Lysis (insert) and Terminator (vector):

 * For Terminator (V): cut with E/X in Buffer2 for 1 hour, and 2 hours (digestion time check)
 * For Lysis (I): cut with E/S in Buffer2 for 1 hour, and 2 hours
 * 1245-1315: QIA PCR purification for the above 4 samples (as MinElute columns not enough).
 * 1315-1415: gel loading and running (1% gel) (4 samples x 3 lanes = 12 lanes)
 * 1330-1400: staining
 * 1400-1500: gel extraction + Nanodrops
 * 1500-1530: ligation
 * 1530-1600: MinElute PCR purification (got 3 columns left before this)
 * 1600-1800: Transformation into competent cells (only used 3ul of ligation mixture, the other 7ul was put in -20 fridge).

Digestional check for E7-empty vector and T7ptag-empty vector "miniprepped" on Saturday
The amounts used for digestional check are: DNA: 2ul EcorI: 0.5ul PstI: 0.5ul Buffer2: 2ul BSA: 0.2ul H2O: 14.8ul Total: 20ul
 * Incubate for 2 hours
 * 1415-1545: gel loading +staining
 * RESULTS:
 * 2 E7 samples only got bands at around 2kb. For each sample, there were 2 separate bands: one at 2kb (which is GFP vector), the other one slightly above 2kb. The latter is expected to be E7 insert (as we cannot find any other possibility for that band). So, as far as we can tell, we've already got E7 successfully ligated into standard empty vector!
 * 1 T7ptag sample got 2kb band (GFP vector) and 2.8kb band (T7ptag). So we also successfully obtained T7ptag in standard plasmid form!!