Team:DTU-Denmark/Notebook/12 August 2013
From 2013.igem.org
12 August 2013
Contents |
lab 208
Main purpose
- PCR reaction in order to amplify backbone pZA21 containing araBAD and RFP
- PCR reaction in order to amplify Nir1 and Nir2 fragments
Who was in the lab
Henrike, Kristian, Gosia
Procedure
PCR mix for backbone
- PCR mix - according to standard protocol.
- Primers - 1an and 1b
- Template - "Ara" sample (plasmid isolation pZA21:araBAD:RFP).
- Program was based on standard program with 65 C of annealing temperature and 4 min of extension time.
- Samples names: 1, 2, 3 and N (negative)
PCR for Nir1 and Nir2
- PCR mix according to standar protocol with changes: addition of DMSO in 3 different final concentrations (2%, 3%, 5%); two different buffers (HF 5x and GC 5x), amount of added water was dependent on volume of added DMSO.
- Primers for Nir1 - 39a, 39b
- Primers for Nir2 - 40a, 40b
- Templates - fragments Nir1 and Nir2 amplified with non-uracil primers, gel purified
- Polymerase x7
- Samples names:
- Nir1, GC buffer, 2% DMSO
- Nir1, GC buffer, 3% DMSO
- Nir1, GC buffer, 5% DMSO
- Nir1, HF buffer, 2% DMSO
- Nir1, HF buffer, 3% DMSO
- Nir1, HF buffer, 5% DMSO
Results
Conclusion
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