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User:University of Washington/5 August 2008
From 2008.igem.org
LuxR from pLac
-I0462 part sequence confirmed. Overnight started of that part for glycerol stock.
-R+E sequence appears inconclusive again, alot of n's present.
-Transformation of second R+E ligation plated succesfully on amp plate. Colony selected, and overnight started for sequencing.
-Overnight of DH5a+LacIq started to make electrocompetent.
MG1655Z1
- Restriction digest minipreped DNA of plasmids in MG1655Z1 (expected no plasmids left after cure) with XbaI.
- After 2 hours, ran gel on the digested DNA together with the non-digest minipreped. The result showed that there were still plasmids left in the strain. In the digested DNA lane, there were bands between 5-6 kb, 10 kb and above.
- Inoculated MG1655Z1 from glycerol stock made from original strain received on Amp plate to check if there was other unknown plasmids.
LuxR from AraC and TetR
- Nanodropped the digested P1010(AC): 63.5 ng/ul, 1.02-260/280, 0.27-260/230; and promoter D29: 26.4 ng/ul, 0.81-260/280, 0.33-260/230.
- Ligation of P1010 on pSB1AC3 and promoter 29.
- Mixed 16.47 ul dH2O + 2 ul T4ligase buffer + 0.32 ul P1010(20 ng) + 0.21 ul D29(~5.5 ng) + 1 ul T4 ligase
- Incubated 10 ul of the reaction in fridge overnight.
- Incubated the other 10 ul of the reaction at room temp 30 mins
- denatured enzyme at 65 degree Celcius 10 mins
- 20 min on filter paper. (note: mistakenly used Scott's cellulose filter paper, barely got anything left on the paper.)
- electroporated whatever was left into 45 ul XL1 Blue and plated on Amp.
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