Team:Groningen/Notebook/31 August 2009

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Wet

GVP Cluster

Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pSB1A2-pLacI-GVP 41.2 1.85 2.18 ? Yes (EcoRI/PstI)
pSB1A2-pBad/araC-GVP 421.7 1.84 2.34 ? Yes (EcoRI/PstI)
pSB1AC3-H 2.43.1 1.86 2.17 All Used Yes (Glycerol Stock)


Restriction for Assembly

The vector pSB1A2 containing the pLacI/GVP and pBad/araC/GVP composite parts were cut with PstI and EcoRI to create correct ends for insert into pSB1AC3, which was also cut with EcoRI and PstI.

Plasmid Amount μL MQ μL Fast digest buffer EcoRI fast digest enzyme XbaI fast digest enzyme SpeI fast digest enzyme PstI fast digest enzyme
pSB1A2-pLacI-GVP 15.0 x 3.0 1.0 x x 1.0
pSB1A2-pBad/araC-GVP 4.0 12.0 3.0 1.0 x x 1.0
pSB1AC3-High 6.0 9.0 3.0 1.0 x x 1.0

Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.

Purification

A "Agarose Gel DNA Extraction Kit" Standard Protocol from Roche Applied Science.

→ In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.

Transporters

'HmtA' The cloning strategy will be changed. First we will make a PCR2 and a PCR1.2 where F2-mut1rc plus Rev-mut2rc will generate a HmtA where we will add a prefix via a PCR with F1-REV. And than ready for cloning.

Metal Accumulation

Vectors

Dry

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