Team:DTU-Denmark/Notebook/25 July 2013
From 2013.igem.org
(9 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
{{:Team:DTU-Denmark/Templates/StartPage|25 July 2013}} | {{:Team:DTU-Denmark/Templates/StartPage|25 July 2013}} | ||
- | =208= | + | 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== | ||
Line 15: | Line 16: | ||
==Procedure== | ==Procedure== | ||
<hr/> | <hr/> | ||
- | |||
- | |||
===gel electrophoresis=== | ===gel electrophoresis=== | ||
Line 25: | Line 24: | ||
* 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=== | + | ===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=== | ===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=== | ||
Line 72: | Line 85: | ||
==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.
Navigate to the Previous or the Next Entry