Team:DTU-Denmark/Notebook/25 July 2013
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(→ON cultures and re-plating of Nir USER transformants from Colony PCR) |
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- | =208= | + | {{:Team:DTU-Denmark/Templates/StartPage|25 July 2013}} |
+ | Navigate to the [[Team:DTU-Denmark/Notebook/24_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_July_2013|Next]] Entry | ||
+ | =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/> | ||
- | |||
- | |||
===gel electrophoresis=== | ===gel electrophoresis=== | ||
===ON cultures and re-plating of Nir USER transformants from Colony PCR=== | ===ON cultures and re-plating of Nir USER transformants from Colony PCR=== | ||
- | Positive Nir USER transformants obtained by 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 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 | + | * 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== | ||
+ | <hr/> | ||
===Gel 1=== | ===Gel 1=== | ||
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===Gel 2=== | ===Gel 2=== | ||
+ | |||
+ | # 1kb ladder | ||
+ | # pZA21 plasmid backbone, USER primers | ||
+ | # pZA21 plasmid backbone, USER primers | ||
+ | # Nir1, extraction PCR, 5uL template | ||
+ | # Nir2, extraction PCR, 5uL template | ||
+ | # Nir whole fragment, extraction PCR, 5uL template | ||
+ | # Nir1, extraction PCR, 10uL template | ||
+ | # Nir2, extraction PCR, 10uL template | ||
+ | # Nir whole fragment, extraction PCR, 10uL template | ||
+ | # Nir 1, 5 uL, U | ||
+ | # Nir1, 10uL, U | ||
+ | # Nir2, 5uL, U | ||
+ | # Nir2, 10uL, U | ||
+ | # Nir 25 colony PCR | ||
+ | # Nir 26 colony PCR | ||
+ | # Nir 27 colony PCR | ||
+ | # Nir 28 colony PCR | ||
+ | # 1kb ladder | ||
+ | |||
+ | [[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. | ||
Navigate to the [[Team:DTU-Denmark/Notebook/24_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_July_2013|Next]] Entry | Navigate to the [[Team:DTU-Denmark/Notebook/24_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_July_2013|Next]] Entry | ||
{{:Team:DTU-Denmark/Templates/EndPage}} | {{:Team:DTU-Denmark/Templates/EndPage}} |
Latest revision as of 22:06, 29 September 2013
25 July 2013
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 kb ladder
- AMO 5uL template, 1
- AMO 5uL template, 2
- AMO 10uL template, 1
- AMO 10uL template, 2
- HAO 5uL template, 1
- HAO 5uL template, 2
- HAO 10uL template, 1
- HAO 10uL template, 2
- restriction analysis cyc EcoRI
- restriction analysis cyc HindIII
- 1 kb ladder
Gel 2
- 1kb ladder
- pZA21 plasmid backbone, USER primers
- pZA21 plasmid backbone, USER primers
- Nir1, extraction PCR, 5uL template
- Nir2, extraction PCR, 5uL template
- Nir whole fragment, extraction PCR, 5uL template
- Nir1, extraction PCR, 10uL template
- Nir2, extraction PCR, 10uL template
- Nir whole fragment, extraction PCR, 10uL template
- Nir 1, 5 uL, U
- Nir1, 10uL, U
- Nir2, 5uL, U
- Nir2, 10uL, U
- Nir 25 colony PCR
- Nir 26 colony PCR
- Nir 27 colony PCR
- Nir 28 colony PCR
- 1kb ladder
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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