Team:DTU-Denmark/Notebook/6 August 2013

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==Procedure==
==Procedure==
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====PCR for AMO====
====PCR for AMO====

Revision as of 18:07, 6 August 2013

6 August 2013

Contents

lab 208


Main purpose


Who was in the lab


Kristian, Gosia, Natalia, Henrike

Procedure


PCR for AMO

Made four reactions using 1 uL respectively 10 uL of culture from the -20 or from the glycerol stock (-80) as template and adjusted volume of water accordingly.

Primers 10a, 10b.

PCR for SPL (synthtic promoter library)

template: pZa21 with RFP, primers: 52a, 52b1, temperature: 50C, time: 2:30


PCR for reference promoter

template: pZa21 with RFP, primers: 52a, 52b2, temperature: 50C, time: 2:30


PCR for araBAD K808000

template: K808000, primers: 12a, 12bn, temperature: 50C, time: 2:30

gel purification

of the Nir1 fragment extraced from P. aeruginosa

plasmid miniprep

HAO in pZA21 from USER cloning was purified for restriction analysis

Results


1% agarose gel

  • 1 kb ladder
  • Nir1 col. T
  • Nir1 col. T
  • Nir2 col. T
  • Nir2 col. T
  • Nir1 frag. T
  • Nir1 frag. T
  • Nir1 col. X7
  • Nir1 col. X7
  • neg
  • Hi
  • Hi
  • DMSO
  • DMSO
  • Nir1
  • Nir1
  • Nir2
  • Nir2
  • 1 kb ladder

2013-08-06 nir.jpg

1% agarose gel

  • 1 kb ladder
  • NirW
  • NirW
  • araBAD biobrick K808000
  • araBAD biobrick K808000
  • SPL in pZA21 containing RFP
  • SPL in pZA21 containing RFP
  • reference promoter in pZA21 containing RFP
  • reference promoter in pZA21 containing RFP
  • neg
  • neg
  • 1 kb ladder

(pic small gel)

Gel result:

  • 1 - ladder
  • 2 - sample nr 1 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
  • 3 - sample nr 2 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
  • 4 - sample nr 3 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
  • 5 - sample nr 4 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
  • 6 - sample nr 5 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
  • 7 - sample nr 6 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
  • 8 - sample nr 7 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
  • 9 - sample nr 8 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
  • 10 - sample nr 9 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
  • 11 - sample nr 10 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
  • 12 - sample nr 11 - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, Phusion polymerase
  • 13 - sample nr 12 - - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, x7 polymerase
  • 14 - sample nr 13 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
  • 15 - sample nr 14 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
  • 16 - sample nr 15 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
  • 17 - sample nr 16 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
  • 18 - ladder

Conclusion


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