Team:Groningen/Notebook/17 August 2009

From 2009.igem.org

Wet

GVP Cluster

→ DONE Restriction for Assembly

→ TODO Gel purification of plasmid

→ TODO Ligation of metal promoter oligo's into vector BBa_J61002

→ TODO Transformation of E.coli TOP10 cells

→ TODO Test promoter/GVP constructs (grow precultures)

→ TODO Grow o.n. cultures to check ligation of pSB3K3-H/L-GVP constructs of last week (3 each)

Restriction for Assembly

The vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high] constitutive promoter was cut with EcoRI and SpeI to create correct ends for insert of metal promoter oligo's.

→ Restriction in this way should cut out the original promoter, but the fragment is small and hard to detect on gel. The different size between double cut and single cut vector is to small to separate, and a bit of luck is needed to avoid self ligation of original promoter into vector.

• 8 μL plasmid in MQ (1.0μg)
• 8 μL MQ (end volume of 20μL)
• 2μL Fast digest buffer
• 1μL PstI fast digest enzyme
• 1μL SpeI fast digest enzyme

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

Purification

→ The 1% agarose MP (1xTBE) gave either a to weak, or to hard gel and new solution was made to be sure of the agarose concentration!

→ After addition of 4μL 6x loading buffer to the restriction solutions the cups were stored on ice until the new gels were ready. The entire contents of the cups were loaded on gel to give a higher concentration of purified plasmid in the end.

[[Image:|250px]]

→ From left to right:1kB ladder,

Concentrations

Ligation

Tranformation

Transporters

Metal Accumulation

Vectors

Dry