Team:DTU-Denmark/Notebook/25 July 2013

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=208=
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{{:Team:DTU-Denmark/Templates/StartPage|25 July 2013}}
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=Lab 208=
<hr/>
<hr/>
==Main purpose==
==Main purpose==
<hr/>
<hr/>
* PCR of AMO and HAO using USER primers and as a template using AMO and HAO extracted from chromosomal DNA with non-uracil primers
* PCR of AMO and HAO using USER primers and as a template using AMO and HAO extracted from chromosomal DNA with non-uracil primers
 +
* PCR of Nir with new primers, template - cells of ''Pseudomonas''
* verification of PCRs
* verification of PCRs
* ON cultures and re-plating of Nir USER transformants from Colony PCR ([[Team:DTU-Denmark/Notebook/17_July_2013#PCR| 17-07-2013]])
* ON cultures and re-plating of Nir USER transformants from Colony PCR ([[Team:DTU-Denmark/Notebook/17_July_2013#PCR| 17-07-2013]])
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==Procedure==
==Procedure==
<hr/>
<hr/>
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===PCR in order to amplify AMO and HAO with USER primers===
 
===gel electrophoresis===
===gel electrophoresis===
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1% agarose gel was loaded with samples as follows:
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===ON cultures and re-plating of Nir USER transformants from Colony PCR===
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Positive Nir USER transformants obtained by colony PCR:
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* Samples 25 and 26 were inoculated in 5 mL LB medium + 30 ug/ml kanamicyn for ON cultures preparation.
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* Samples 25, 26 and 27 were re-plated in LB agar + 30 ug/ml kanamicyn
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# 1 kb ladder
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===PCR to amplify AMO and HAO with USER primers===
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# AMO 5uL template, 1
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# AMO 5uL template, 2
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# AMO 10uL template, 1
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# AMO 10uL template, 2
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# HAO 5uL template, 1
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# HAO 5uL template, 2
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# HAO 10uL template, 1
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# HAO 10uL template, 2
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# restriction analysis cyc EcoRI
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# restriction analysis cyc HindIII
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# 1 kb ladder
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===ON cultures and re-plating of Nir USER transformants from Colony PCR===
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* AMO - primers 17a, 17b, 54 C annealing, 3 min of extension
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2 positive Nir USER transformants obtained by colony PCR were inoculated in 5 mL LB medium + 30 ug/ml kanamicyn for ON cultures preparation
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* HAO - primers 18a, 18b, 59 C annealing, 3 min extension
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Samples: 1,2 for AMO contain linear template - fragments extracted from chromosomal DNA with non-USER primers, sample 3 contains 5 uL of liquid culture of ''Nitrosomonas europaea''
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Samples 4,5 for HAO contain linear template - fragments extracted from chromosomal DNA with non-USER primers, sample 6 contains 5 uL of liquid culture of ''Nitrosomonas europaea''
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===PCR to amplify Nir===
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New primers which do not contain uracil, polymerase Phusion is used. Template is 5 uL of liquid culture of ''Pseudomonas''.
 +
 
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Nir is being amplified in two fragments.
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Samples are as follows:
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* 1.1 and 1.2 - First part of Nir, primers 41a, 41b, 63C annealing T, 9 min elongation
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* 2.1 and 2.2 - Second part of Nir, primers 42a, 42b, 63C annealing T, 9 min elongation
==Results==
==Results==
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<hr/>
===Gel 1===
===Gel 1===
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# 1 kb ladder
# 1 kb ladder
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[[File:2013-07-25_AMO_HAO_and_restriction_cyc_EcoRI_HindIII.jpg|600px]]
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===Gel 2===
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# 1kb ladder
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# pZA21 plasmid backbone, USER primers
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# pZA21 plasmid backbone, USER primers
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# Nir1, extraction PCR, 5uL template
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# Nir2, extraction PCR, 5uL template
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# Nir whole fragment, extraction PCR, 5uL template
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# Nir1, extraction PCR, 10uL template
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# Nir2, extraction PCR, 10uL template
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# Nir whole fragment, extraction PCR, 10uL template
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# Nir 1, 5 uL, U
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# Nir1, 10uL, U
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# Nir2, 5uL, U
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# Nir2, 10uL, U
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# Nir 25 colony PCR
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# Nir 26 colony PCR
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# Nir 27 colony PCR
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# Nir 28 colony PCR
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# 1kb ladder
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[[File:2013-07-25_AMO_HAO_and_restriction_cyc_EcoRI_HindIII.jpg|600px]]
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[[File:2013-07-25 Nir with new primers.jpg|600px]]
==Conclusion==
==Conclusion==
 +
The PCR of AMO and HAO with User endings was unsucessful. Restriction analysis of the plasmid containing cytochromes did not show the expected result.
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Latest revision as of 22:06, 29 September 2013

25 July 2013

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Contents

Lab 208

Main purpose

  • PCR of AMO and HAO using USER primers and as a template using AMO and HAO extracted from chromosomal DNA with non-uracil primers
  • PCR of Nir with new primers, template - cells of Pseudomonas
  • verification of PCRs
  • ON cultures and re-plating of Nir USER transformants from Colony PCR ( 17-07-2013)

Who was in the lab

Kristian, Gosia, Julia

Procedure

gel electrophoresis

ON cultures and re-plating of Nir USER transformants from Colony PCR

Positive Nir USER transformants obtained by colony PCR:

  • Samples 25 and 26 were inoculated in 5 mL LB medium + 30 ug/ml kanamicyn for ON cultures preparation.
  • Samples 25, 26 and 27 were re-plated in LB agar + 30 ug/ml kanamicyn

PCR to amplify AMO and HAO with USER primers

  • AMO - primers 17a, 17b, 54 C annealing, 3 min of extension
  • HAO - primers 18a, 18b, 59 C annealing, 3 min extension

Samples: 1,2 for AMO contain linear template - fragments extracted from chromosomal DNA with non-USER primers, sample 3 contains 5 uL of liquid culture of Nitrosomonas europaea Samples 4,5 for HAO contain linear template - fragments extracted from chromosomal DNA with non-USER primers, sample 6 contains 5 uL of liquid culture of Nitrosomonas europaea

PCR to amplify Nir

New primers which do not contain uracil, polymerase Phusion is used. Template is 5 uL of liquid culture of Pseudomonas.

Nir is being amplified in two fragments. Samples are as follows:

  • 1.1 and 1.2 - First part of Nir, primers 41a, 41b, 63C annealing T, 9 min elongation
  • 2.1 and 2.2 - Second part of Nir, primers 42a, 42b, 63C annealing T, 9 min elongation

Results

Gel 1

  1. 1 kb ladder
  2. AMO 5uL template, 1
  3. AMO 5uL template, 2
  4. AMO 10uL template, 1
  5. AMO 10uL template, 2
  6. HAO 5uL template, 1
  7. HAO 5uL template, 2
  8. HAO 10uL template, 1
  9. HAO 10uL template, 2
  10. restriction analysis cyc EcoRI
  11. restriction analysis cyc HindIII
  12. 1 kb ladder

2013-07-25 AMO HAO and restriction cyc EcoRI HindIII.jpg

Gel 2

  1. 1kb ladder
  2. pZA21 plasmid backbone, USER primers
  3. pZA21 plasmid backbone, USER primers
  4. Nir1, extraction PCR, 5uL template
  5. Nir2, extraction PCR, 5uL template
  6. Nir whole fragment, extraction PCR, 5uL template
  7. Nir1, extraction PCR, 10uL template
  8. Nir2, extraction PCR, 10uL template
  9. Nir whole fragment, extraction PCR, 10uL template
  10. Nir 1, 5 uL, U
  11. Nir1, 10uL, U
  12. Nir2, 5uL, U
  13. Nir2, 10uL, U
  14. Nir 25 colony PCR
  15. Nir 26 colony PCR
  16. Nir 27 colony PCR
  17. Nir 28 colony PCR
  18. 1kb ladder

2013-07-25 Nir with new primers.jpg

Conclusion

The PCR of AMO and HAO with User endings was unsucessful. Restriction analysis of the plasmid containing cytochromes did not show the expected result.

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