Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/03: Difference between revisions

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<br>  
Amount of DNA used for each reaction according to their concentrations:  
Amount of DNA used for each reaction w/r to their concentrations:  
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| align="center" style="background:#f0f0f0;"|'''RhlR'''
| align="center" style="background:#f0f0f0;"|'''RhlR'''
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| 3.7||3.6||5.9
| 3.7||3.6||5.9
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<br><br>
<b>3. Ligate 3:1 ratio of insert:bb </b>
Amounts of DNA used were based on vector/insert size. <br>
Vector is ~3,200bp. Transcription factor inserts are ~851bp. <br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Volume (uL)'''
| align="center" style="background:#f0f0f0;"|''''''
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| ||EsaR||RhlR||LuxR ||Vector-only
|-
| Insert DNA (40ng)||6uL ||6uL ||6uL||0uL
|-
| Vector DNA (50ng)||1.52uL||1.52uL||1.52uL||1.52uL
|-
| 10x Buffer||2uL||2uL||2uL||2uL
|-
| Ligase||1uL||1uL||1uL||1uL
|-
| Water ||9.48uL||9.48uL||9.48uL||15.48uL
|-
| Total ||20uL||20uL||20uL||20uL
|}
|}


Revision as of 17:42, 3 July 2015

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06/3/2015

Performed ligations of modular receiver vector + RhlR, LuxR, and EsaR regulator ORFs.
1. Digest 1ug of modular receiver vector

Reagent Vol (uL)
DNA 1.45 (1ug at 691.8ng/uL)
EcoRI 1
XbaI 1
Phosphotase 1
FD Buffer 3
Water 22.55
Total: 30

Incubate at 37°C for 15 minutes.
Heat inactivate at 80°C for 5 minutes; 65°C for 20 minutes.

2. Digest PCR amplified, then purified, regulator ORFs with EcoRI and XbaI.

Reagent Vol (uL)
DNA variable; 200ng
EcoRI 1
XbaI 1
FD Buffer 3
Water 21.3
Total: 30


Amount of DNA used for each reaction w/r to their concentrations:

RhlR EsaR LuxR
3.7 3.6 5.9



3. Ligate 3:1 ratio of insert:bb Amounts of DNA used were based on vector/insert size.
Vector is ~3,200bp. Transcription factor inserts are ~851bp.

' Volume (uL) '
EsaR RhlR LuxR Vector-only
Insert DNA (40ng) 6uL 6uL 6uL 0uL
Vector DNA (50ng) 1.52uL 1.52uL 1.52uL 1.52uL
10x Buffer 2uL 2uL 2uL 2uL
Ligase 1uL 1uL 1uL 1uL
Water 9.48uL 9.48uL 9.48uL 15.48uL
Total 20uL 20uL 20uL 20uL


PCR amplify inducible promoters for: Aub, Bta, Cer, and Exp. (Bta, Cer, and Exp seemed to be running low; unsure if these will work.




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