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Solution Informationhelp
Enzyme: DNA recombination protein RecA
inhibitor: BDBM31772
substrate: n/a
 
Solution Type: Aqueous
pH at Preparation: n/a
Temp. Prep.: n/a
Comments: GFP-RecA Splicing Assay in Dose 1. Dispense positive control Dispense 100 nL of positive control ZnCl2(60mM) using Combi nl in respective wells according to plate design to 1536-well assay ready plates (Aurora 00027830) that contain 22.5 nL/well of 10 mM compound in 8 doses with 3-fold dilution(generated by Echo acoustic dispenser) 2. Dispense GFP-RecA Intein Add 3 uL/well of GFP-recA intein(2uM) using Combi nl(Thermo), incubate at room temperature for 30 minutes. 3. Dispense TCEP to initiate the reaction Add 3uL/well of TCEP(2mM) with Combi nL (Thermo), incubate at room temperature for ~18 hours 4. Centrifuge Centrifuge the plates at 1000 rpm x1min 5. Read on Envision for Fluorescence(Ex.405nm/Em.510nm) Solutions: Intein buffer 20 mM NaPi 7.0 500 mM NaCl 500 mM L-Arginine.HCL 0.01 % Tritin-100 1 % DMSO ZnCl2 (in H2O, Sigma 229997-50G) 60 mM ZnCl2 TCEP (in intein buffer, Sigma 646547) 2 mM TCEP GFP-RecA intein (in intein buffer, provided by Henry Paulus's lab)
 
 

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Last update November 1, 2007
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