Team:DTU-Denmark/Notebook/30 August 2013

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30 August 2013

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Contents

lab 208


Main purpose


  • User reaction
  • PCR to amplify HAO and cycAX USER fragments

Who was in the lab


Procedure


USER reaction

Redid USER reaction for ligating our inserts into the Biobrick backbone. There was no cycAX fragment left.

HAO in pSB1C3 AMO in pSB1C3

PCR to amplify HAO and cycAX USER fragments

Made 6 reactions for each, using either the extraction PCR fragment or the glycerol stock as template

label cyc gly D cyc gly % cyc gly 1uL D cyc ext D cyc ext % cyc ext 1uL D
template glycerol stock glycerol stock glycerol stock extraction PCR fragment extraction PCR fragment extraction PCR fragment
DMSO 1,5 - 1,5 1,5 - 1,5
MgCl2 - - 1 - - 1
MQ 1 2,5 - 1 2,5 -
label HAO gly D HAO gly % HAO gly 1uL D HAO ext D HAO ext % HAO ext 1uL D
template glycerol stock glycerol stock glycerol stock extraction PCR fragment extraction PCR fragment extraction PCR fragment
DMSO 1,5 - 1,5 1,5 - 1,5
MgCl2 - - 1 - - 1
MQ 1 2,5 - 1 2,5 -

Program:

temperature time cycles
98C 2:00 -
98C 0:20 36
annealing temperature 1:00 36
72C extension time 36
72C 5:00 -
10C hold -

HAO - 59C, 3:00

cycAX - 55C, 1:30

Results


Gel

  • 1 kb ladder
  • psB1C3 for Nir, 2% DMSO
  • psB1C3 for Nir, 2% DMSO, 1 uL 50mM MgCl2
  • psB1C3 for Nir, 4% DMSO
  • psB1C3 for Nir, 4% DMSO, 1 uL 50mM MgCl2
  • psB1C3 for Nir, 1M Betaine
  • purification of psB1C3 USER fragment to make Biobricks (escaped from well -> Ethanol?)
  • purification of HAO fragment (escaped from well -> Ethanol?)
  • purification of AMO fragment (escaped from well -> Ethanol?)
  • purification of psB1C3 USER fragment to make Biobricks (used more loading buffer)
  • purification of HAO fragment (used more loading buffer)
  • purification of AMO fragment (used more loading buffer)
  • purification of pZA21::ara(tight)
  • purification of pZA21::ara(tight) with endings for Nir
  • 1 kb ladder

2013-08-30 psbnir puri verification2.jpg

Conclusion


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