Team:DTU-Denmark/Notebook/16 August 2013

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{{:Team:DTU-Denmark/Templates/StartPage|16 August 2013}}
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=lab 208=
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=Lab 208=
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==Main purpose==
==Main purpose==
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*Gradient PCR for Nir2 USER
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*BioBrick construction
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*SPL project
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*Preparation for sequencing
==Who was in the lab==
==Who was in the lab==
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===Gradient PCR for Nir2 USER===
===Gradient PCR for Nir2 USER===
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Set up new gradient PCR for Nir2 USER to run in 223. Reaction mix without MgCl2 but using DMSO and GC-buffer.
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Set up new gradient PCR for Nir2 USER to run in 223. Reaction mix without MgCl<sub>2</sub> but using DMSO and GC-buffer.
*template: Nir2 extraction fragment
*template: Nir2 extraction fragment
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*buffer: GC
*buffer: GC
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Gradient went from 60C to 72C in 12 steps.
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Gradient went from 60C to 72 <sup>o</sup>C in 12 steps.
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Note: Redid this PCR with X7 instead of PHUSION polymerase.
<u>pSB1C3</u>:
<u>pSB1C3</u>:
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Done with primerpair 54 as a two step PCR program with 98C in 10 sec. and 72C for 1:10 min looping 35 times.
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Done with primer pair 54.
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{| class="wikitable" style="text-align: right"
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! temperature !! time !! cycles
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|  98C || 2:00 || -
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|  98C || 0:10 || 36
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|  58C || 0:20 || 36
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|  72C || 0:35 || 36
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|  72C || 10:00 || -
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|  10C || hold || -
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===SPL project===
===SPL project===
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==Conclusion==
==Conclusion==
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Navigate to the [[Team:DTU-Denmark/Notebook/15_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/17_August_2013|Next]] Entry
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The amplification of the User-ready backbone was sucessfull, but we have to redo the PCR for the signal peptides and the cytochromes. Nir2 with USEr endings was sucessfully amplified.
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Navigate to the [[Team:DTU-Denmark/Notebook/15_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/19_August_2013|Next]] Entry
{{:Team:DTU-Denmark/Templates/EndPage}}
{{:Team:DTU-Denmark/Templates/EndPage}}

Latest revision as of 11:59, 4 October 2013

16 August 2013

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Contents

Lab 208

Main purpose

  • Gradient PCR for Nir2 USER
  • BioBrick construction
  • SPL project
  • Preparation for sequencing

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 72 oC 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 -

Note: Redid this PCR with X7 instead of PHUSION polymerase.

pSB1C3:

Done with primer pair 54.

temperature time cycles
98C 2:00 -
98C 0:10 36
58C 0:20 36
72C 0:35 36
72C 10:00 -
10C hold -

SPL project

Picked single colonies of araBAD ref, constitutive SPL and constitutive ref transformants and replated them on plates sprayed with arabinose 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

2013-08-16 nir2 user gradient.jpg

Samples 5-10 and 12 were cut out for gel purification.

Conclusion

The amplification of the User-ready backbone was sucessfull, but we have to redo the PCR for the signal peptides and the cytochromes. Nir2 with USEr endings was sucessfully amplified.

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