Monday 21 July

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).

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!!