Endy:Notebook/In vitro Flipping assay

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Project Description/Abstract
Our goal is to set up in vitro assays to quantitatively measure the kinetic of DNA inversion by recombinases.

Existing in vitro recombination assays

 * Bxb1 LxR recombination, in-vitro, Fluorescein+BHQ Ghosh-PNAS-2008
 * -see figure 6
 * -from text: "We observed substantial quenching with suicide substrate 2 when antiparallel alignment of the sites places the fluorescein label and the quencher on the same side of the protein–DNA complex"

=Flipping Assay=

FRET/quencher based assay

 * a linear piece of DNA contain a fluorophore and a quencher flanking a recombination site. State 0= quenched state 1= bright.
 * How do we build the test construct ? check with IDT
 * Which are the best quencher/ fluorophore pairs?
 * Places to start looking for examples:
 * -Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols
 * -Molecular Beacons: Signalling Nucleic Acid Probes, Methods, and Protocols


 * Which devices do we need to perform the experiment?
 * one of the fluorescence detectors at the high-throughput center might be suitable

Quantitative PCR assay

 * Principles of quantitative PCR starting reference


 * Which devices do we need to perform the reading (spectro, QPCR machine?), where can we find them? (guess we have some in the lab)

Restriction assay
Classic, but pretty sure to get raw information the target plasmid is digested by enzymes in and outside the flippee.

=Recombinase purification=
 * Protocols for purifying integrases.
 * which recombinases to start? Bxb1 seems a good candidate.

To order:

Novagen Rosetta strain BL21 DE3

EMD chemicals Rosetta™ 2 Competent Cells	71402-3	0.4 ml	Y		$88.00

=Experimental procedure=
 * detailled protocol on how to perform the assay

=Using the data for modeling= What parameters should be taken into account to incorporate these datas in a computational model?