Difference between revisions of "Part:BBa K4604025:Design"
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===Cloning strategy=== | ===Cloning strategy=== | ||
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Plasmid piG_23 (<a class="link" href="https://parts.igem.org/Part:BBa_K4604024">BBa_K4604024</a>) 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. </html> | Plasmid piG_23 (<a class="link" href="https://parts.igem.org/Part:BBa_K4604024">BBa_K4604024</a>) 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. </html> | ||
Latest revision as of 23:38, 11 October 2023
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.