Team:DTU-Denmark/Notebook/16 August 2013

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16 August 2013

Contents

lab 208


Main purpose


Who was in the lab


Kristian, Julia, Henrike

Procedure


Gradient PCR for Nir2 USER

Set up new gradient PCR for Nir2 USER to run in 223. Reaction mix without MgCl2 but using DMSO and GC-buffer.

  • template: Nir2 extraction fragment
  • primers: 40a, 40b
  • additives: 5% DMSO
  • buffer: GC

Gradient went from 60C to 72C in 12 steps.

sample # 1 2 3 4 5 6 7 8 9 10 11 12
temperature 60.2 60.5 61.6 63.0 64.2 65.4 66.6 67.6 68.9 70.2 71.6 72

PCR for BioBrick construction

PCR amplified the constructs from Hello World and cytochromes and pSB1C3, which will be ligated together into biobricks.

constructs:

  • GC-buffer, 5%DMSO, 2uL 50mM MgCl2
  • primers: 53a, 53b
  • templates: TAT3-2, Sec2, cycAX

Tried two different annealing temperatures. Program:

temperature time cycles
98C 2:00 -
98C 0:10 36
60C/56C 0:45 36
72C 0:45 36
72C 5:00 -
10C hold -

pSB1C3:

Done with primerpair 54 as a two step PCR program with 98C in 10 sec. and 72C for 1:10 min looping 35 times.

SPL project

Picked single colonies of araBAD SPL transformants and replated them to prepare for Biolector experiment to measure the strength of the expression.

Preparation for sequencing

Diluted miniprepped plasmids down to 100 ng/uL. Have to redo cycAx in pZA21 purification because it was diluted too much.

Results


Gel on today's PCR for the Biobrick project

  • 1 kb ladder
  • Sec2 on 60C
  • TAT3-2 on 60C
  • cycAX on 60C
  • Sec2 on 56C
  • TAT3-2 on 56C
  • cycAX on 56C
  • negative for construct parts
  • pSB1C3 backbone
  • pSB1C3 backbone
  • pSB1C3 backbone
  • pSB1C3 backbone
  • pSB1C3 backbone
  • pSB1C3 backbone
  • 1 kb ladder

2013-08-16 biobrick psb1c3.jpg

Gel on screening PCR to amplify Nir2 USER

  • 1 kb ladder
  • Nir2 USER, sample 1
  • Nir2 USER, sample 2
  • Nir2 USER, sample 3
  • Nir2 USER, sample 4
  • Nir2 USER, sample 5
  • Nir2 USER, sample 6
  • Nir2 USER, sample 7
  • Nir2 USER, sample 8
  • Nir2 USER, sample 9
  • Nir2 USER, sample 10
  • Nir2 USER, sample 11
  • Nir2 USER, sample 12
  • 1 kb ladder

Conclusion


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