Difference between revisions of "Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/04/29"

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(Autocreate 2014/04/29 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs)
 
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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>}}
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|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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==mm/dd/yyyy==
 
==mm/dd/yyyy==
* Insert content here...
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Ran PCR products from yesterday on a gel. 5ul of reactions 2-9, all 30 of 1 so I can gel extract because there should be two bands and I only want one.  
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<br>
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DpnI digest PCR reactions 2-7 and 9 with positive and negative controls (didn't run reaction 8 because I checked the gel early and saw that there was no DNA there).
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<br>
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Controls
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Positive control was a miniprepped plasmid at 100ng/ul concentration.
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
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| align="center" style="background:#f0f0f0;"|''''''
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|-
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| Thing||Positive ctrl||Negative ctrl
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|-
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| H2O||12||12
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|-
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| Green digest buffer||2||2
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|-
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| pTet mCh||5||5
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|-
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| DpnI||1||0
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|}
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<br><br>
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Thing'''
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| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
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|-
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| Digest buffer||4
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|-
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| PCR rxn||25
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|-
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|H2O||1
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|-
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| DpnI||1
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|}
  
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Incubated at 37°C for 55 minutes, sat on the bench for 3 minutes, heat inactivated at 80 for 5 minutes.<br>
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Cleaned using zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions.
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<br>
 +
Gel extracted PCR reaction 1. Saw a smaller band above, think I got the correct band.
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<br>
 +
{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''PCR  reaction'''
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| align="center" style="background:#f0f0f0;"|'''Description'''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|'''260'''
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| align="center" style="background:#f0f0f0;"|'''280'''
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| align="center" style="background:#f0f0f0;"|'''260/280'''
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| align="center" style="background:#f0f0f0;"|'''ng/µL'''
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|-
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| 1||Rec bb||Gel extracted||0.02||0.01||2||20.289
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|-
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| 2||pLux-GFP||DpnI digested, column purified||0.047||0.024||2||47.148
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|-
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| 3||pRhl-GFP||DpnI digested, column purified||0.05||0.025||2.004||50.466
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|-
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| 4||pEsa-GFP||DpnI digested, column purified||0.032||0.015||2.117||32.1
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|-
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| 5||pLac-LuxR||DpnI digested, column purified||0.046||0.024||1.935||45.771
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|-
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| 6||pLac-RhlR||DpnI digested, column purified||0.06||0.035||1.692||59.88
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|-
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| 7||pLac-RhlR||DpnI digested, column purified||0.041||0.019||2.2||41.438
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|-
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| 9||pLac-EsaR||DpnI digested, column purified||0.038||0.018||2.135||37.847
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|-
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|
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|}
  
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Didn't have enough of the backbone so started another PCR. 1-4 are int receiver vector P112 P123, 5-6 are purified PCR product of receiver backbone P112 P123.
 
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|}
 
|}
  
 
__NOTOC__
 
__NOTOC__

Latest revision as of 22:56, 26 September 2017

Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

mm/dd/yyyy

Ran PCR products from yesterday on a gel. 5ul of reactions 2-9, all 30 of 1 so I can gel extract because there should be two bands and I only want one.
DpnI digest PCR reactions 2-7 and 9 with positive and negative controls (didn't run reaction 8 because I checked the gel early and saw that there was no DNA there).
Controls Positive control was a miniprepped plasmid at 100ng/ul concentration.

' Volume (ul) '
Thing Positive ctrl Negative ctrl
H2O 12 12
Green digest buffer 2 2
pTet mCh 5 5
DpnI 1 0



Thing Volume (ul)
Digest buffer 4
PCR rxn 25
H2O 1
DpnI 1

Incubated at 37°C for 55 minutes, sat on the bench for 3 minutes, heat inactivated at 80 for 5 minutes.
Cleaned using zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions.
Gel extracted PCR reaction 1. Saw a smaller band above, think I got the correct band.

PCR reaction Description ' 260 280 260/280 ng/µL
1 Rec bb Gel extracted 0.02 0.01 2 20.289
2 pLux-GFP DpnI digested, column purified 0.047 0.024 2 47.148
3 pRhl-GFP DpnI digested, column purified 0.05 0.025 2.004 50.466
4 pEsa-GFP DpnI digested, column purified 0.032 0.015 2.117 32.1
5 pLac-LuxR DpnI digested, column purified 0.046 0.024 1.935 45.771
6 pLac-RhlR DpnI digested, column purified 0.06 0.035 1.692 59.88
7 pLac-RhlR DpnI digested, column purified 0.041 0.019 2.2 41.438
9 pLac-EsaR DpnI digested, column purified 0.038 0.018 2.135 37.847

Didn't have enough of the backbone so started another PCR. 1-4 are int receiver vector P112 P123, 5-6 are purified PCR product of receiver backbone P112 P123.