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Team:DTU-Denmark/Notebook/11 July 2013
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*PCR with USER primers to [[Team:DTU-Denmark/Notebook/11_July_2013#Combine_Nir_operon_with_pZA21|combine Nir operon with pZA21]] | *PCR with USER primers to [[Team:DTU-Denmark/Notebook/11_July_2013#Combine_Nir_operon_with_pZA21|combine Nir operon with pZA21]] | ||
| - | *PCR with USER primer to test the activity of the promoter AraB.[[Team:DTU-Denmark/Notebook/11_July_2013#PCR_for_promoter_test|PCR for promoter test]] | + | *PCR with USER primer to test the activity of the promoter AraB. [[Team:DTU-Denmark/Notebook/11_July_2013#PCR_for_promoter_test|PCR for promoter test]] |
==Who was in the lab== | ==Who was in the lab== | ||
Revision as of 13:53, 11 July 2013
Contents |
208
Main purpose
- PCR with USER primers to combine Nir operon with pZA21
- PCR with USER primer to test the activity of the promoter AraB. PCR for promoter test
Who was in the lab
Ariadni,Henrike,Julia,Jakob
Procedure
Combine Nir operon with pZA21
Following Mathildes Protocol for DpnI digestion of our linear pZA21 w. Uracil incerts
Mastermix for 7 PCR tubes, Using the mastermix protocol we got from Mathilde.
triplets of each of the two Nir Operon extracts that we have made yesterday, including one negative control:
- Nir 2a(I,II,III)
- Nir 2b(I,II,III)
Using the following PCR program:
| time | Temp C | |
|---|---|---|
| Denaturing | 2 min | 98 |
| start of loop | 10 sec | 98 |
| annealing | 30 sec | 60 |
| elongation | 9 min | 72 |
| goto start of loop 35 times | ||
| hold | 10 |
PCR for promoter test
We want to test the inducible AraB promoter using plasmid pZA21 without its native promoter and RFP gene. We performed 3 different PCR reactions using user primers. Samples are named:
- 1,2,3 -> pZA21 with no native promoter - Primers 13a and 13b, template pZA21 miniprep
- 4,5,6 -> RFP - Primers 14a and 14b, template RFP in pZE21 miniprep
- 7,8,9 -> AraB promoter - Primers 12a and 12b, template biobrick K808000
Settings for PCR of 1,2,3
| Temperature (oC) | Time (min) | Rounds |
|---|---|---|
| 98 | 2:00 | 1 |
| 98 | 0:20 | 35 |
| 58 | 0:45 | 35 |
| 72 | 2:00 | 35 |
| 72 | 5:00 | 1 |
| 10 | ∞ | - |
Settings for PCR of 4-9
| Temperature (oC) | Time (min) | Rounds |
|---|---|---|
| 98 | 2:00 | 1 |
| 98 | 0:20 | 35 |
| 61 | 0:45 | 35 |
| 72 | 1:00 | 35 |
| 72 | 5:00 | 1 |
| 10 | ∞ | - |
