Team:DTU-Denmark/Notebook/23 August 2013

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23 August 2013

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Contents

Lab 208


Main purpose


  • PCR to isolate Biobricks
  • PCR to make the constitutive reference promoter
  • colony PCR on Nir transformants

Who was in the lab


Kristian, Henrike

Procedure


PCR Biobrick Isolation

Used new primers to isolate Hello World constructs. Used HF buffer and tested three different concentrations of DMSO: 0%, 2% and 5%.

Primers: 56a, 56b

template: TAT2-1 (miniprep)

program:

temperature time cycles
98C 2:00 -
98C 0:20 36
56C 0:30 36
72C 0:45 36
72C 5:00 -
10C hold -

PCR constitutive reference promoter

Got new primers for the constitutive reference promoter since the old reverse primer (52b2) had a mistake. Repeated reaction set-up and program from the 13.08. Used GC buffer, 5% DMSO and 2uL 50 mM MgCl2.

primers: 52a, 52bn

template: pZA21::RFP


program:

temperature time cycles
98C 2:00 -
98C 0:20 36
58.1C 1:00 36
72C 3:00 36
72C 5:00 -
10C hold -

Colony PCR on Nir transformants

Used Q5 premix from NEB.

Reagent volume (in uL)
Q5 master mix 12,5
FW primer 3
RV primer 3
template 1
MilliQ 5,5

primers: 45a, 45b

program:

temperature time cycles
98C 10:00 -
98C 0:10 36
56C 0:30 36
72C 0:45 36
72C 5:00 -
10C hold -

Results


Gel electrophoresis

  • 1 kb ladder
  • Sec2 Biobrick
  • Sec2 Biobrick
  • Sec2 neg
  • col AMO 1
  • col AMO 2
  • col AMO 3
  • col AMO 4
  • col AMO 5
  • col AMO 6
  • col AMO 7
  • col AMO 8
  • col AMO 9
  • col AMO 10
  • 1 kb ladder

2013-08-23 bb sec amo colony.jpg

  • 1 kb ladder
  • col Nir
  • constitutive reference promoter
  • constitutive reference promoter
  • Biobrick Isolation TAT2-1 negative
  • Biobrick Isolation TAT2-1 2% DMSO
  • Biobrick Isolation TAT2-1 5% DMSO
  • Biobrick Isolation TAT2-1 w/o DMSO
  • 1 kb ladder

Conclusion


The transformation of AMO into pZA21::ara was successful, all tested colonies have the insert.

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