Difference between revisions of "Part:BBa K4604019:Design"
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A stop codon was added to terminate translation after MazF.
 
A stop codon was added to terminate translation after MazF.
  
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===Cloning of piG_03===
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  Plasmid piG_02b (<a class="link" href="https://parts.igem.org/Part:BBa_K4604018">BBa_K4604018</a>) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 62°C and an elongation time of 2 minutes. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto a 1% 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.
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Revision as of 19:22, 10 October 2023


piG_03 (tetR_RiboK12_mazF)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 710
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 883
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1518


Design Notes

A stop codon was added to terminate translation after MazF.

Cloning of piG_03

Plasmid piG_02b (BBa_K4604018) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 62°C and an elongation time of 2 minutes. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto a 1% 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

This BioBrick is BBa_K4604018 without the fluorescent protein mTurquoise, cloned via Gibson Assembly.

References