Team:DTU-Denmark/Notebook/30 August 2013

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30 August 2013

Contents

lab 208


Main purpose


Who was in the lab


Procedure


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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