Since the colony-PCR (26.05.14) was negative and the Gibson-Assembly of linearized pMAD-fla with cup1-1 showed no results the pET24d-fla construct was linearized with SpeI.
Restriction |
Volume [µl] |
pET24d-fla (98.8 ng/µl) |
8 |
SpeI |
1 |
CutSmart-Buffer (10x) |
2 |
H2O |
9 |
Total Volume |
20 |
The restriction was incubated at 37°C for 1 h.
The restriction mix was purified with the Gel Ex Kit. The yield was too low (c = 3.1 ng/µl) to use it for a Gibson-Assembly, so the rest of the pET24d-fla was restricted with SpeI.
Restriction |
Volume [µl] |
pET24d-fla (98.8 ng/µl) |
18,5 |
SpeI |
1 |
CutSmart-Buffer (10x) |
2,5 |
H2O |
3 |
Total Volume |
25 |
The restriction was incubated at 37°C for 1h.
The restriction mix was purified with the Gel Ex Kit (c = 20.1 ng/µl), which was enough for the Gibson-Assembly. 4µl (80 ng) of this restricted plasmid were mixed with 1 µl of the cup1-1 fragment (9.6 ng/µl). These 5 µl were inserted into the prepared 15µl Gibson-mix. The reaction was incubated at RT for 1 min and then at 50°C for 1 h. An extra Gibson-reaction was carried out as a control with just the linearized pET24d-fla without the cup1-1.
Competent E. coli XLI-Blue were transformed with the whole Gibson-reactions. They were plated on LB-Kan an incubated at 37°C overnight.