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

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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

1:12:13:1Bb ctrl
Insert0.511.50
Vector0.660.660.660.66
2x ligation buffer5555
Ligase1111
Water32.523.5

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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