Team:DTU-Denmark/Notebook/6 August 2013

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{{:Team:DTU-Denmark/Templates/StartPage|6 August 2013}}
{{:Team:DTU-Denmark/Templates/StartPage|6 August 2013}}
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Navigate to the [[Team:DTU-Denmark/Notebook/5_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/7_August_2013|Next]] Entry
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=lab 208=
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=Lab 208=
<hr/>
<hr/>
==Main purpose==
==Main purpose==
<hr/>
<hr/>
 +
* Extraction PCR for AMO
 +
* PCR for SPL (synthetic promoter library)
 +
* PCR for reference promoter
 +
* PCR for araBAD biobrick K808000
 +
* PCR to extract Nir2 from ''P. aeruginosa''
 +
* PCR for araBAD and SPL with DMSO
 +
* PCR on Nir1 with MgCl2 gradient
 +
* Gel purification of Nir1 (from ''P. aeruginosa'')
 +
* Plasmid isolation: HAO in pZA21 from USER cloning.
==Who was in the lab==
==Who was in the lab==
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<hr/>
<hr/>
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====PCR for AMO====
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===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.
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.
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Primers 10a, 10b.
Primers 10a, 10b.
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====PCR for SPL (synthtic promoter library)====
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===PCR for SPL (synthetic promoter library)===
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template: pZa21 with RFP, primers: 52a, 52b1, temperature: 50C, time: 2:30
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template: pZA21 with RFP, primers: 52a, 52b1, temperature: 50C, time: 2:30
-
====PCR for reference promoter====
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===PCR for reference promoter===
-
template: pZa21 with RFP, primers: 52a, 52b2, temperature: 50C, time: 2:30
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template: pZA21 with RFP, primers: 52a, 52b2, temperature: 50C, time: 2:30
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====PCR for araBAD K808000====
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===PCR for araBAD K808000===
template: K808000, primers: 12a, 12bn, temperature: 50C, time: 2:30
template: K808000, primers: 12a, 12bn, temperature: 50C, time: 2:30
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===gel purification===
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===PCR to extract Nir2 from P. aeruginosa===
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of the Nir1 fragment extraced from P. aeruginosa
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===PCR for araBAD and SPL with DMSO===
 +
Template and primers as previous.
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===plasmid miniprep===
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===PCR on Nir1 with MgCl2 gradient===
 +
Used previous isolation from a gel purification to make PCR for amplification of the strand. Used 5% DMSO as additive. The PCR was done with primer pair 41 and with x7 polymerase at 55C annealing and 5:00 min extension. Also we used 20 sec as denaturing time instead of the normal 10.
 +
The magnesium gradient was as follow:
 +
*0
 +
*1uL MgCl2 2mM per 50uL reaction
 +
*5uL MgCl2 2mM per 50uL reaction
 +
*20uL MgCl2 2mM per 50uL reaction
 +
 
 +
===Gel purification===
 +
 
 +
of the Nir1 fragment extracted from ''P. aeruginosa''. Nanodrop: 8ng/uL
 +
 
 +
===Plasmid miniprep===
HAO in pZA21 from USER cloning was purified for restriction analysis. Nanodrop measurement: 180ng/uL
HAO in pZA21 from USER cloning was purified for restriction analysis. Nanodrop measurement: 180ng/uL
Line 42: Line 64:
<hr/>
<hr/>
 +
===Gel 1===
1% agarose gel
1% agarose gel
* 1 kb ladder
* 1 kb ladder
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* Nir1 col. T
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* Nir1 col. Taq
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* Nir1 col. T
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* Nir1 col. Taq
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* Nir2 col. T
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* Nir2 col. Taq
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* Nir2 col. T
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* Nir2 col. Taq
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* Nir1 frag. T
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* Nir1 frag. Taq
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* Nir1 frag. T
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* Nir1 frag. Taq
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* Nir1 col. X7
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* Nir1 col. x7
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* Nir1 col. X7
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* Nir1 col. x7
* neg
* neg
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* Hi
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* Hi spec. buffer
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* Hi
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* Hi spec. buffer
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* DMSO
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* DMSO  
* DMSO  
* DMSO  
* Nir1
* Nir1
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[[File:2013-08-06 nir.jpg| 600px]]
[[File:2013-08-06 nir.jpg| 600px]]
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Nir1 was purified.
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 +
===Gel 2===
1% agarose gel
1% agarose gel
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* 1 kb ladder
* 1 kb ladder
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(pic small gel)
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[[File:2013-08-06 small gel.jpg|600px]]
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===Gel result:===
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===Gel 3===
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* 1 - ladder
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1% agarose gel
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* 2 - sample nr 1 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
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-
* 3 - sample nr 2 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
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* 1 kb ladder
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* 4 - sample nr 3 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
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* AMO, 1uL of -20 culture as template
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* 5 - sample nr 4 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
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* AMO, 10uL of -20 culture as template
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* 6 - sample nr 5 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
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* AMO, 1uL of glycerol stock as template
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* 7 - sample nr 6 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
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* AMO, 10uL of glycerol stock as template
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* 8 - sample nr 7 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
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* neg from AMO PCR
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* 9 - sample nr 8 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
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* 1 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
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* 10 - sample nr 9 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
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* 2 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
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* 11 - sample nr 10 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
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* 3 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
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* 12 - sample nr 11 - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, Phusion polymerase
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* 4 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
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* 13 - sample nr 12 - - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, x7 polymerase
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* 5 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
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* 14 - sample nr 13 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
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* 6 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
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* 15 - sample nr 14 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
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* 1 kb ladder
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* 16 - sample nr 15 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
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* 17 - sample nr 16 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
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[[File:2013-08-06 AMO and spl.jpg| 600px]]
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* 18 - ladder
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 +
AMO was gel purified.
==Conclusion==
==Conclusion==
<hr/>
<hr/>
 +
*Nir was obtained from colony PCR and purified.
 +
*AMO was extracted and gel purified.
 +
Navigate to the [[Team:DTU-Denmark/Notebook/5_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/7_August_2013|Next]] Entry
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{{:Team:DTU-Denmark/Templates/EndPage}}
{{:Team:DTU-Denmark/Templates/EndPage}}

Latest revision as of 22:09, 29 September 2013

6 August 2013

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Contents

Lab 208

Main purpose

  • Extraction PCR for AMO
  • PCR for SPL (synthetic promoter library)
  • PCR for reference promoter
  • PCR for araBAD biobrick K808000
  • PCR to extract Nir2 from P. aeruginosa
  • PCR for araBAD and SPL with DMSO
  • PCR on Nir1 with MgCl2 gradient
  • Gel purification of Nir1 (from P. aeruginosa)
  • Plasmid isolation: HAO in pZA21 from USER cloning.

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 (synthetic 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

PCR to extract Nir2 from P. aeruginosa

PCR for araBAD and SPL with DMSO

Template and primers as previous.

PCR on Nir1 with MgCl2 gradient

Used previous isolation from a gel purification to make PCR for amplification of the strand. Used 5% DMSO as additive. The PCR was done with primer pair 41 and with x7 polymerase at 55C annealing and 5:00 min extension. Also we used 20 sec as denaturing time instead of the normal 10. The magnesium gradient was as follow:

  • 0
  • 1uL MgCl2 2mM per 50uL reaction
  • 5uL MgCl2 2mM per 50uL reaction
  • 20uL MgCl2 2mM per 50uL reaction

Gel purification

of the Nir1 fragment extracted from P. aeruginosa. Nanodrop: 8ng/uL

Plasmid miniprep

HAO in pZA21 from USER cloning was purified for restriction analysis. Nanodrop measurement: 180ng/uL

Results

Gel 1

1% agarose gel

  • 1 kb ladder
  • Nir1 col. Taq
  • Nir1 col. Taq
  • Nir2 col. Taq
  • Nir2 col. Taq
  • Nir1 frag. Taq
  • Nir1 frag. Taq
  • Nir1 col. x7
  • Nir1 col. x7
  • neg
  • Hi spec. buffer
  • Hi spec. buffer
  • DMSO
  • DMSO
  • Nir1
  • Nir1
  • Nir2
  • Nir2
  • 1 kb ladder

2013-08-06 nir.jpg

Nir1 was purified.

Gel 2

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

2013-08-06 small gel.jpg

Gel 3

1% agarose gel

  • 1 kb ladder
  • AMO, 1uL of -20 culture as template
  • AMO, 10uL of -20 culture as template
  • AMO, 1uL of glycerol stock as template
  • AMO, 10uL of glycerol stock as template
  • neg from AMO PCR
  • 1 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
  • 2 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
  • 3 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
  • 4 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
  • 5 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
  • 6 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
  • 1 kb ladder

2013-08-06 AMO and spl.jpg

AMO was gel purified.

Conclusion

  • Nir was obtained from colony PCR and purified.
  • AMO was extracted and gel purified.

Navigate to the Previous or the Next Entry

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