Part:BBa_K4604025:Design
piG_23b (tetR_riboRBS_mazF)
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 701
Illegal XbaI site found at 616 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 701
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 701
Illegal BglII site found at 710
Illegal BamHI site found at 1153
Illegal XhoI site found at 1162 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 701
Illegal XbaI site found at 616 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 701
Illegal XbaI site found at 616 - 1000COMPATIBLE WITH RFC[1000]
Cloning strategy
Plasmid piG_23 (BBa_K4604024) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 57°C and an elongation time of 3 minutes. A DpnI digest was done at 37°C overnight, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. Gibson Assembly was used according to the protocol to assemble the plasmid. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were taken for the next steps. Since this was only a change of the RBS, screening was not possible. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.