Team:DTU-Denmark/Notebook/13 August 2013

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13 August 2013

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Contents

lab 208


Main purpose


Who was in the lab


Kristian, Henrike, Gosia

Procedure


PCR (promoter library)

PCR to amplify USER fragments of the constitutive SPL, the constitutive reference promoter and the arabinose reference promoter.

Used 5% DMSO, 2 uL of mM MgCl2 and GC-buffer.

constitutive SPL

  • template: pZA21::RFP
  • primers: 52a, 52b1
  • annealing temp: 58.1C
  • elongation time: 3:00

constitutive reference

  • template: pZA21::RFP
  • primers: 52a, 52b2
  • annealing temp: 58.1C
  • elongation time: 3:00

Wanted to make negative for the two above reactions but accidentally put the template in the master mix.

arabinose reference

  • template: pZA21:araBAD:RFP (labeled Ara)
  • primers: 51a,51b1
  • annealing temp: 60.3C
  • elongation time: 4:00

program (used for all 3 reactions):

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

PCR Nir2

PCR to gain a bigger amount of the extraction fragment of Nir2

sample nr. buffer  %DMSO program
1
2
3
4
5
6
7 ramp
8 ramp
9 ramp
10 ramp
11 ramp
12 ramp

Gel purification

Gel purified araBAD SPL from yesterdays screening PCR

Results


Gel of SLP screening PCR products (yesterday)

Each gel is representing one row; from left to right and up to down is row A to H:

Gel of todays PCR reactions

  • 1kb ladder
  • Nir2 PCR - sample 1
  • Nir2 PCR - sample 2
  • Nir2 PCR - sample 3
  • Nir2 PCR - sample 4
  • Nir2 PCR - sample 5
  • Nir2 PCR - sample 6
  • Nir2 PCR - sample 7
  • Nir2 PCR - sample 8
  • Nir2 PCR - sample 9
  • Nir2 PCR - sample 10
  • Nir2 PCR - sample 11
  • Nir2 PCR - sample 12
  • constitutive SPL 1
  • constitutive SPL 2 (duplicate)
  • constitutive promoter reference 1
  • constitutive promoter reference 2 (duplicate)
  • constitutive promoter reference 3 (very small sample, there was almost nothing left in the master mix)
  • 1kb ladder

2013-08-13 nir2 const spl ref.jpg

Conclusion


SLP screening PCR

The PCR run with condition E (5% DMSO, 2mM MgCl2 with GC buffer) had the highest flexibility; it yields the expected product at different annealing temperatures: 58.4 C, 59.6, C 60.6 C, 61.9 C, 63.2 C, 64.6 C, 65 C.

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