Endy:Notebook/In vitro Flipping assay

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<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">Endy:Notebook/In vitro Flipping assay</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>

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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[1]
-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

  • 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?

Ideas for an in vivo assay

Notes

  • Place some notes here for visitors
  • Example: This project is currently on hold until further notice.

References

  1. Ghosh P, Bibb LA, and Hatfull GF. Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading. Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3238-43. DOI:10.1073/pnas.0711649105 | PubMed ID:18299577 | HubMed [Ghosh-PNAS-2008]
Recent changes

25 September 2017

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