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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==06/3/2015==
==07/3/2015==


<font color = purple>  
<font color = purple>  
Performed ligations of modular receiver vector + RhlR, LuxR, and EsaR regulator ORFs. <br>
<h4 font color = purple> Ligate modular receiver vector (MRV) + RhlR, LuxR, and EsaR. </h4>
<b>1. Digest 1ug of modular receiver vector </b>  
<b>1. Digest 1ug MRV </b>  
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Reagent'''
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Incubate at 37°C for 15 minutes. <br>
:Incubate at 37°C for 15 minutes. <br>
Heat inactivate at 80°C for 5 minutes; 65°C for 20 minutes. <br><br>
:Heat inactivate at  
:*80°C for 5 minutes
:*65°C for 20 minutes


<b> 2. Digest PCR amplified, then purified, regulator ORFs with EcoRI and XbaI. </b>
<b> 2. Digest PCR amplified and purified, regulators w/ E+X. </b>
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Reagent'''
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<br>  
<br>  
Amount of DNA used for each reaction according to their concentrations:  
Amount of DNA used for each reaction w/r to their concentrations:  
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''RhlR'''
| align="center" style="background:#f0f0f0;"|'''RhlR'''
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| align="center" style="background:#f0f0f0;"|'''LuxR'''
| align="center" style="background:#f0f0f0;"|'''LuxR'''
|-
|-
| 3.7||3.6||5.9
| 3.7uL||3.6uL||5.9uL
|}
|}
<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;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume (uL)'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| ||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
|}
<br>
<b> 4. Transformation with DH5αT cells </b> <br>
:10uL of each ligation reaction were used in the transformation.
----




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




Latest revision as of 01:02, 27 September 2017

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07/3/2015

Ligate modular receiver vector (MRV) + RhlR, LuxR, and EsaR.

1. Digest 1ug MRV

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 and purified, regulators w/ E+X.

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
3.7uL 3.6uL 5.9uL



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.
Reagent 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


4. Transformation with DH5αT cells

10uL of each ligation reaction were used in the transformation.


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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