Team:TU Darmstadt/safety/Labjournal

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<font size="8" color="#F0F8FF" face="Arial regular">Labbook |</font>
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font>
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For the construction of pSB1C3-petZ we isolated the pezT gene via PCR from the gBlocks C1, C2 and C3 following this protocol:
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For the construction of pSB1C3-pezT we isolated the pezT gene via PCR from the gBlocks C1, C2 and C3 following this protocol:
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Latest revision as of 01:56, 5 October 2013







Lab book | Materials | Protocols



Labjournal

gBlocks assembly

We designed the light induced kill switch based on the pDawn plasmid. We ordered 10 gBlock fragments coding for our plasmid from IDT. We assembeld the fragments according to the following protocol:

Assembly PCR with 10 gBlocks
Fig. 1:
M: GeneRuler DNA Ladder Mix (Thermo Sc.)

1: Assembly PCR. The signal at 5 kbps is our construct.

  • Reconstitute the gBlock fragments in 10 µl TE buffer
  • Use 1 µl of each gBlock fragment for a PCR with the Q5 Polymerase
  • Perform the PCR reaction with primers coding for the prefix and suffix and an annealing temperature of 55 °C
    (30 cycles)
  • Load an 0.8% agarose gel with 5 µl of PCR reaction (total volume 50 µl) and perform a DNA gel electrophoresis
  • Cut the other 45 µl with the restriction enzymes EcoRI and PstI (10 U each) for 1 h at 37 °C
  • Ligate 5 µl of the reaction mix with 50 ng EcoRI/PstI restricted and purified pSB1C3 over night at 16 °C
    For Ligation use the T4-Ligase and fresh T4-Ligase Buffer with ATP
  • After heat-inactivation of the ligase by 80 °C for 15 min use 2 µl of the mix for heat-shock transformation
  • Plate out the transformation mix on black petridishies with LB-cmp media



safety




Construction of pSB1C3-pezT

For the construction of pSB1C3-pezT we isolated the pezT gene via PCR from the gBlocks C1, C2 and C3 following this protocol:

Zeta_toxin_pcr

Fig. 2: M: 1kbp Ladder (Promega)
1: PCR isolated pezT gene. The pezT gene shows a signal at 700 bps
2: Unrestricted pSB1C3
3: pSB1C3 cut by PstI and EcoRI. The empty plasmid shows a signal at 1.3 kbps

  • Use the primer pair Pre-PezT/For-PezT and an annealing temperature of 55 °C. For the isolation
    PCR follow the Q5 PCR protocol from NEB Biolabs.
  • Use 5 µl for the control gel electrophoresis (1.2% agarose) and clean the rest of the reaction with
    the Wizard SV Gel and PCR Clean-Up System from Promega.
  • Restrict 500 ng of the PCR product and 1 µg of pSB1C3 with EcoRI and PstI (10 U each) at 37 °C for 30 min.
  • Perform the ligation with an molar ratio of 3:1 (insert:vector).
    For Ligation use the T4-Ligase and fresh T4-Ligase Buffer with ATP
  • Transform 2 µl of the ligation mix into E. coli DH5α.
  • Plate out the transformation on petri dishes with LB-cmp media.


safety




Conclusion
  • The results from the gBlock assembly PCR (Fig. 1) could not be reproduced.
  • The gBlock assembly PCR resulted in the right signal (Fig. 2). Nevertheless multiple transformations failed.








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