Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/06/28: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|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>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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*Digest LasR_MRV with BbsI  
*Digest LasR_MRV with BbsI  
*{| {{table}}
 
! Reagent !! Volume (uL)
:{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume (uL)'''
|-
| DNA (500ng)||1.5
|-
| 10x Green FD Buffer||2.0
|-
| BbsI||1.0
|-
| Roche SAP||1.0
|-
| Water||14.5
|-
| Total||20
|}
 
:Incubate at 37°C for 10-20 minutes
:Heat inactivate rSAP at 75°C for 2 minutes
:Heat inactivate BbsI at 65°C for 20 minutes
 
*Digest pCer and pBta3R
:{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume (uL)'''
|-
| DNA (500ng)||12
|-
| 10x Green FD Buffer||3
|-
| EcoRI || 1
|-
| SpeI || 1
|-
| Water||13
|-
| Total||30
|}
:Incubate at 37°C for 10-20 minutes
:PCR purify
 
 
 
*PCR pExp and pLas
:{| {{table}}
| align="center" style="background:#f0f0f0;"|''' Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume (uL)'''
|-
| F Primer||1
|-
| R Primer||1
|-
| DNA Template||1
|-
|-
| DNA (500ng) || 1.5
| Taq 2x MM||25
|-
|-
| 10x Green FD Buffer || 2.0
| Water||22
|-
|-
| BbsI || 1.0
| Total||50
|}
 
:Thermocycler:
:{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Temp (°C)'''
| align="center" style="background:#f0f0f0;"|'''Length (s)'''
|-
|-
| Roche SAP || 1.0
| 95||30
|-
|-
| Water || 14.5
| 95||15
|-
|-
| Total || 20
| 60-70||30
|-
| 72||15
|-
| 72||5m
|}
|}
:Extension for 15 minutes because of small size. Gradient of 60-70°C because pExp needs a higher annealing temperature to increase specificity; pLas uses 60°C to increase yield because it's plasmid is simple and does not contain similar binding sites. pExp was annealed at 66-70°C.


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Latest revision as of 01:50, 27 September 2017

Project name Main project page
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06/28/2016

  • Digest LasR_MRV with BbsI
Reagent Volume (uL)
DNA (500ng) 1.5
10x Green FD Buffer 2.0
BbsI 1.0
Roche SAP 1.0
Water 14.5
Total 20
Incubate at 37°C for 10-20 minutes
Heat inactivate rSAP at 75°C for 2 minutes
Heat inactivate BbsI at 65°C for 20 minutes
  • Digest pCer and pBta3R
Reagent Volume (uL)
DNA (500ng) 12
10x Green FD Buffer 3
EcoRI 1
SpeI 1
Water 13
Total 30
Incubate at 37°C for 10-20 minutes
PCR purify


  • PCR pExp and pLas
Reagent Volume (uL)
F Primer 1
R Primer 1
DNA Template 1
Taq 2x MM 25
Water 22
Total 50
Thermocycler:
Temp (°C) Length (s)
95 30
95 15
60-70 30
72 15
72 5m
Extension for 15 minutes because of small size. Gradient of 60-70°C because pExp needs a higher annealing temperature to increase specificity; pLas uses 60°C to increase yield because it's plasmid is simple and does not contain similar binding sites. pExp was annealed at 66-70°C.


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