Part:BBa_K4604021:Design
piG_10a (tetR_bluB_riboK12_mazF)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 710
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1603
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2238
Design Notes
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Cloning of piG_10a
Plasmid piG_03 (BBa_K4604019 was used as a backbone and amplified according to the protocol for the Q5 polymerase with an annealing temperature 56°C and elongation time of 4 minutes. For the PCR of the insert (bluB) we also used the general protocol for the Q5 polymerase with an annealing temperature of 65°C and an elongation time of 1 minute. A Dpn1 digest was done at 37°C for an hour, 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 screened by colony PCR. 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.
Source
piG_03 (BBa_K4604019) was used as a backbone, bluB (BBa_K4604005) was inserted via Gibson Assembly.