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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
- -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"
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)
Classic, but pretty sure to get raw information
the target plasmid is digested by enzymes in and outside the flippee.
- Protocols for purifying integrases.
- which recombinases to start? Bxb1 seems a good candidate.
Novagen Rosetta strain BL21 DE3
EMD chemicals Rosetta™ 2 Competent Cells 71402-3 0.4 ml Y $88.00
- 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
- Place some notes here for visitors
- Example: This project is currently on hold until further notice.
- 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 |