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Team:DTU-Denmark/Notebook/13 August 2013
From 2013.igem.org
13 August 2013
Contents |
lab 208
Main purpose
Who was in the lab
Kristian, Henrike, Gosia
Procedure
PCR (promoter library)
PCR to amplify USER fragments of the constitutive SPL, the constitutive reference promoter and the arabinose reference promoter.
Used 5% DMSO, 2 uL of mM MgCl2 and GC-buffer.
constitutive SPL
- template: pZA21::RFP
- primers: 52a, 52b1
- annealing temp: 58.1C
- elongation time: 3:00
constitutive reference
- template: pZA21::RFP
- primers: 52a, 52b2
- annealing temp: 58.1C
- elongation time: 3:00
arabinose reference
- template: pZA21:araBAD:RFP (labeled Ara)
- primers: 51a,51b1
- annealing temp: 60.3C
- elongation time: 4:00
program (used for all 3 reactions)
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:20 | 36 |
| annealing temp | 1:00 | 36 |
| 72C | extension time | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
PCR Nir2
PCR to gain a bigger amount of the extraction fragment of Nir2
| sample nr. | buffer | %DMSO | program |
|---|---|---|---|
| 1 | |||
| 2 | |||
| 3 | |||
| 4 | |||
| 5 | |||
| 6 | |||
| 7 | ramp | ||
| 8 | ramp | ||
| 9 | ramp | ||
| 10 | ramp | ||
| 11 | ramp | ||
| 12 | ramp |
Gel purification
Gel purified araBAD SPL from yesterdays screening PCR
Results
Gel of SLP screening PCR products (yesterday)
Each gel is representing one row; from left to right and up to down is row A to H:
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Conclusion
SLP screening PCR
The PCR run with condition E (5% DMSO, 2mM MgCl2 with GC buffer) had the highest flexibility; it yields the expected product at different annealing temperatures: 58.4 C, 59.6, C 60.6 C, 61.9 C, 63.2 C, 64.6 C, 65 C.
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