New England Biolabs, M0249L, RecA

RecA is necessary for genetic recombination reactions involving DNA repair and UV-induced mutagenesis Related Categories ssDNA Binding Proteins,, DNA Repair Enzymes and Structure-specific Endonucleases Specification Reaction Conditions 1X Rec A Reaction Buffer Incubate at 37°C 1X Rec A Reaction Buffer 70 mM Tris-HCl 10 mM MgCl2 5 mM DTT (pH 7.6 @ 25°C) Usage Concentration 2 mg/ml Storage Buffer 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes FAQ Q: What buffer should be used with RecA Protein? A: The supplied RecA buffer can be used to form stable triple helixes when supplemented with ATP gamma-S. ATP gamma-S is not supplied. Sigma product (A1388) ATP gamma-S can be used. A buffer similar to several published in the scientific literature, 25 mM Tris-acetate (pH 7.8), 4 mM Mg+2 acetate, 0.3 mM ATP gamma-S, 0.4 mM DTT, has been successfully used with the RecA functional assay. Q: Is magnesium required for triple strand formation using RecA Protein? A: Yes. Q: Is ATP gamma-S required for triple strand formation using RecA Protein? A: Yes. ATP gamma-S at a concentration of 0.3 mM will stabilize the triple helix. ATP gamma-S is not supplied. Sigma product (A1388) ATP gamma-S can be used. Q: Is the structure of the target DNA important when using RecA Protein? A: Yes, supercoiled plasmids allow a stable triple-helix to form (1). (1) Zhumabayeva, B., Chenchik, A., and Siebert, P. D., Biotechniques 27:834-845, 1999. Q: How much RecA Protein should be added to the single stranded DNA to coat it? A: The suggested ratio is 3 moles bases to 1 mole RecA. Q: What length single stranded oligo should be used with RecA Protein? A: The length of the oligo should be at least 30 and can be as long as 60 nucleotides long. The site being blocked should be in the center of the oligo.