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

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Incubate at RT for 35min<br>
Incubate at RT for 35min<br>
Add 40ul DH5αT cells to entire rxn<br>
Add 40ul DH5αT cells to entire rxn<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>
'''GG PCR'''<br>
'''GG PCR'''<br>
Ran PCR from 10/8 on gel<br>
Ran PCR from 10/8 on gel<br>

Revision as of 15:59, 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

Part Enzymes Concentration Length
J06702 E/X 75.2 ng/ul 3024bp
Gblock E/S 2.6ng/ul ~80bp

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

1:1 2:1 3:1 Bb ctrl
Insert 0.5 1 1.5 0
Vector 0.66 0.66 0.66 0.66
2x ligation buffer 5 5 5 5
Ligase 1 1 1 1
Water 3 2.5 2 3.5

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



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