New England Biolabs, M0264L, RecJf

Related Categories Exonucleases and Non-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to produce 0.05 nmol TCA soluble deoxyribonucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X NEBuffer 2 with 1.5 µg sonicated single-stranded [ 3 H]-labeled E. coli DNA. Reaction Conditions 1X NEBuffer™ 2 Incubate at 37°C 1X NEBuffer™ 2 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 1 mM DTT (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes FAQ Q: What is the activity of RecJf in the NEBuffers? A: NEBuffer 1: 20% NEBuffer 2: 100% NEBuffer 3: 100% NEBuffer 4: 100% Q: Can DNA be blunted using RecJf? A: No, short ends 4 bp or less are not good substrates. Use DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) to fill in 5' overhangs (also called 3' recessed ends) and chew back 3' overhangs or use Mung Bean Nuclease (NEB# M0250) to chew back 3' or 5' overhangs. Note: 3' overhangs can not be filled in. Q: What is the difference between RecJf and Exonuclease I (NEB# M0293)? A: RecJf works 5' to 3' unlike Exonuclease I which works 3' to 5'. Q: Can RecJf be heat inactivated? A: Yes. Heat inactivate at 65°C for 20 minutes. Q: Will RecJF work in rCutSmart buffer? A: Yes, RecJF will work in rCutSmart buffer. To see its % functional activity in rCutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart.