07/13/2015
LasR
- pLas has been double digested with EcoRI and SpeI, then heat inactivated using an annealing oligos protocol.
- LasR + MRV has been digested with BbsI and gel-extracted for a gel purification.
- Gel-purify LasR + MRV using Sigma Aldrich Gel Extraction Kit
- Ligate pLas and LasR + MRV
| Reagent
|
Vol (uL)
|
| Backbone |
2
|
| Insert |
1
|
| 2x Lig Buffer |
5
|
| T4 Ligase |
1
|
| Water |
1
|
| Total |
10
|
- Transform with 50μL of DH5αT
- In the backbone-only control, no insert was added; volume of water increased to make up for insert volume.
LuxR
- pLux oligos have been annealed
- 1uL each (top and bottom) at 100μM concentrations
- Modular receiver vector was digested with BbsI, then PCR purified
- Ligate pLux and MRV:
| Reagent
|
Vol (uL)
|
| Backbone |
2.7
|
| Insert |
1
|
| 2x Lig Buffer |
5
|
| T4 Ligase |
1
|
| Water |
1.3
|
| Total |
11
|
- Transform with 50μL of DH5αT
- In the backbone-only control, no insert was added; volume of water increased to make up for insert volume.
|
Project name
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