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

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'''Cloning'''
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'''Cloning'''<br>
Cut 10ul J06702 (131) with E/X in 20ul reaction<br>  
Cut 10ul J06702 (131) with E/X in 20ul reaction<br>  
Cut Gblock pLac-RBS with E/S<br>
Cut Gblock pLac-RBS with E/S<br>
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heat shock at 42°C for 30s<br>
heat shock at 42°C for 30s<br>
ice 2min<br><br>
ice 2min<br><br>
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'''GG PCR'''
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'''GG PCR'''<br>
Ran PCR from 10/8 on gel<br>
Ran PCR from 10/8 on gel<br>
Reporter (pLux) primers worked, no background<br>
Reporter (pLux) primers worked, no background<br>

Revision as of 18:57, 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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