Difference between revisions of "Part:BBa K4604025:Design"

 
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<partinfo>BBa_K4604025 short</partinfo>
  
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<partinfo>BBa_K4604025 SequenceAndFeatures</partinfo>
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===Cloning strategy===
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>html>
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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>

Revision as of 23:37, 11 October 2023

piG_23b (tetR_riboRBS_mazF)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 701
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 1000
    COMPATIBLE WITH RFC[1000]


Cloning strategy

>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>