Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/10/09

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(Autocreate 2013/10/09 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs)
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'''Cloning'''
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Cut 10ul J06702 (131) with E/X in 20ul reaction<br>
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Cut Gblock pLac-RBS with E/S<br>
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Used the FD buffer that doesn't have dye<br>
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Cut for 15 min at 37deg<br>
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purified both with Zymo OligoClean & Concentrator, eluted in 15ul water<br>
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''realized later that this won't work for the vector because uncut vector will be purified with cut vector. led to very high background''<br>
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{| {{table}}
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|-
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|Part||Enzymes||Concentration||Length
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|-
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|J06702|| E/X||75.2 ng/ul||3024bp
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|-
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|Gblock||E/S||2.6ng/ul||~80bp
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|-
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|}
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Insert:backbone ratios 1:1, 2:1, 3:1
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{| {{table}}
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|-
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| ||1:1||2:1||3:1||Bb ctrl
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|-
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|Insert||0.5||1||1.5||0
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|-
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|Vector||0.66||0.66||0.66||0.66
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|-
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|2x ligation buffer||5||5||5||5
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|-
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|Ligase||1||1||1||1
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|-
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|Water||3||2.5||2||3.5
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|-
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}}
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Incubate at RT for 35min<br>
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Add 40ul DH5αT cells to entire rxn<br>
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Incubate on ice for 30 mins<br>
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heat shock at 42°C for 30s<br>
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ice 2min<br><br>
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'''GG PCR'''
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Ran PCR from 10/8 on gel<br>
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Reporter (pLux) primers worked, no background<br>
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Sender did not work. Retried with I13522-2

Revision as of 17:57, 15 October 2013

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mm/dd/yyyy

Cloning Cut 10ul J06702 (131) with E/X in 20ul reaction
Cut Gblock pLac-RBS with E/S
Used the FD buffer that doesn't have dye
Cut for 15 min at 37deg
purified both with Zymo OligoClean & Concentrator, eluted in 15ul water
realized later that this won't work for the vector because uncut vector will be purified with cut vector. led to very high background

PartEnzymesConcentrationLength
J06702 E/X75.2 ng/ul3024bp
GblockE/S2.6ng/ul~80bp

Insert:backbone ratios 1:1, 2:1, 3:1

}} Incubate at RT for 35min
Add 40ul DH5αT cells to entire rxn
Incubate on ice for 30 mins
heat shock at 42°C for 30s
ice 2min

GG PCR Ran PCR from 10/8 on gel
Reporter (pLux) primers worked, no background
Sender did not work. Retried with I13522-2
1:12:13:1Bb ctrl
Insert0.511.50
Vector0.660.660.660.66
2x ligation buffer5555
Ligase1111
Water32.523.5


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