Team:DTU-Denmark/Notebook/25 August 2013

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25 August 2013

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Contents

Lab 208


Main purpose


Who was in the lab


Procedure


Colony PCR

Performed colony PCR to confirm insert for HAO, AMO, cycAX and Nir transformants. Used Q5 premix with the following reaction mix:

compound amount
Q5 mix 25 uL
FW primer 3 uL
RV primer 3 uL
template 1 uL
MilliQ 18 uL

Template was made by resuspending 1 culture in 100uL MilliQ.

program:

temperature time cycles
98C 10:00 -
98C 0:10 36
annealing temperature 0:30 36
72C 0:20 36
72C 5:00 -
10C hold -

details (primers, temp, expected fragment length):

  • cycAX - FW_1, RV_2, 61C, 749bp
  • HAO - FW_2, RV_3, 64C, 751bp
  • AMO - FW_2, RV_3, 63C, 750bp
  • Nir - FW_2, RV_3, 64C, 733bp
  • Nir - FW_5, RV_6, 71C, 737bp

Glucose plates

Remade USER ligation and transformation of HAO and cycAX onto plates containing a surplus of glucose. This should reduce the leakiness of the araBAD promoter and improve the health of the transformants before induction so we can recover more colonies.

Results


Conclusion


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