Brandon:Protocols/enzyme rxn buffers
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enzyme reaction buffer compositions[edit]
DNase I
NEB 1X DNase I Reaction Buffer: 10 mM Tris-HCl 2.5 mM MgCl2 0.5 mM CaCl2 pH 7.6 @ 25°C epicentre DNase I 10X Reaction Buffer: 100 mM Tris-HCl (pH 7.5) 25 mM MgCl2 5 mM CaCl2 fermentas Storage Buffer 50 mM Tris-HCl (pH 7.5) 10 mM CaCl2 and 50% (v/v) glycerol 10X Reaction Buffer with MgCl2 100 mM Tris-HCl (pH 7.5 at 25°C) 25 mM MgCl2 1 mM CaCl2.
Magnesium Fragmentation Buffer
NEB 1X NEBNext RNA Fragmentation Reaction Buffer: 40 mM Tris-OAc 100 mM KOAc 30 mM Mg(OAc)2 pH 8.3 @ 25°C NEBNext® RNA Fragmentation Stop Solution (10X) (Store at -20°C or 4°C) (#E6187A: 0.4 ml) 1X NEBNext RNA Fragmentation Stop Solution: 50 mM EDTA
Rnase III
NEB Storage Conditions: 10 mM Tris-HCl 50 mM NaCl 1 mM dithiothreitol 0.5 mM EDTA 50% glycerol pH 8.0 @ 25°C 1X NEBNext RNase III Reaction Buffer: 10 mM Tris-HCl 10 mM Mg(Cl)2 1 mM DTT 60 mM NaCl pH 8.3 @ 25°C
MMLV Reverse transcriptase
promega 1X buffer 50mM Tris-HCl (pH 8.3 at 25°C) 40mM KCl 7mM MgCl2 10mM DTT 0.1mg/ml BSA, 0.5mM [3H]dTTP 0.025mM oligo(dT) 0.25mM poly(A) 0.01% NP-40.
NEB MMLV RT Optimal activity is seen at a Mg2+ concentration of 3 mM. at 10 mM Mg2+, activity drops to 60% The salt optimum is tight around 75 mM (1) SDS, DMSO, high salt, or EDTA in the nucleic acid template preparation can inhibit the reaction Storage Conditions: 50 mM Tris-HCl 150 mM NaCl 1 mM Dithiothreitol 0.1 mM EDTA 50% Glycerol 0.1% NP-40 pH 7.6 @ 25°C reaction buffer 1X M-MuLV Reverse Transcriptase Reaction Buffer: 50 mM Tris-HCl 75 mM KCl 3 mM MgCl2 10 mM Dithiothreitol pH 8.3 @ 25°C
Invitrogen 5X First-Strand Buffer 250 mM Tris-HCl (pH 8.3 at room temperature) 375 mM KCl 15 mM MgCl2 10 mM DTT (2 uL of 100 mM DTT) Storage buffer 20 mM Tris-HCl (pH 7.5) 100 mM NaCl 0.1 mM EDTA 1 mM DTT 0.01% (v/v) NP-40 50% (v/v) glycerol
Clontech 5X first stand buffer 250 mM Tris-HCl (pH 8.3 at room temperature) 375 mM KCl 30 mM MgCl2 20 mM DTT (suggest 20 mM, used 10 mM in our reactions, 2 uL of 100 mM DTT in 20 uL rxn)
T7/T3 RNA Polymerase
invitrogen storage buffer 20 mM Tris-HC1 (pH 7.5) 0.1 M NaCl 0.1 mM EDTA 1 mM DTT 50% (v/v) glycerol 0.01% (w/v) Triton® X-100 reaction buffer 5X T3/T7 Buffer 0.2 M Tris-HCl (pH 8.0) 40 mM MgCl2 10 mM spermidine-(HCl)3 125 mM NaCl
Clontech Storage conditions: 20 mM potassium phosphate (pH 7.9) 100 mM NaCl 1 mM DTT 0.1 mM EDTA 50% glycerol. 10X Reaction Buffer 400 mM Tris-HCl (pH 8.0) 80 mM MgCl2 20 mM spermidine 50 mM DTT
NEB Storage Conditions: 50 mM Tris-HCl 100 mM NaCl 20 mM 2-Mercaptoethanol 1 mM EDTA 50% Glycerol 0.1% Triton X-100 pH 7.9 @ 25°C 1X RNAPol Reaction Buffer: 40 mM Tris-HCl 6 mM MgCl2 10 mM Dithiothreitol 2 mM spermidine pH 7.9 @ 25°C
Poly(A) polymerase
Enzymatics Supplied in 25 mM Tris-HCl 500 mM NaCl 1mM MgCl2 0.1 mM DTT 0.1 mM EDTA 50% glycerol pH 8.0 @ 25°C 10X Poly(A) Polymerase Reaction Buffer (B7460): 500 mM Tris-HCl 2.5 M NaCl 100 mM MgCl2 pH 7.9 @ 25°C
NEB Works in MMLV buffer. recommended by NEB to used MMLV buffer to carry out polyA reaction, then add needed components (primers etc) for MMLV reaction. Reaction Conditions: 1X Poly(A) Polymerase Reaction Buffer Supplemented with 1 mM ATP Incubate at 37°C Storage Conditions: 20 mM Tris-HCl 300 mM NaCl 1 mM DTT 1 mM EDTA 50% Glycerol 0.1% Triton X-100 1X Poly(A) Polymerase Reaction Buffer: 50 mM Tris-HCl 250 mM NaCl 10 mM MgCl2 pH 7.9 @ 25°C
T4 DNA Polymerase
NEB Storage Conditions: 100 mM KPO4 1 mM Dithiothreitol 50% Glycerol pH 6.5 @ 25°C 1X NEBuffer 2: 10 mM Tris-HCl 50 mM NaCl 10 mM MgCl2 1 mM Dithiothreitol pH 7.9 @ 25°C
transposome reaction mixture
Nextera 5x Tagmentation reaction buffer 50 mM Tris-OAc pH 8.0 25 mM Mg(OAc)2
ampligase
NEB 2X Quick Ligation Reaction Buffer: 132 mM Tris-HCL 20 mM MgCl2 2 mM dithiothreitol 2 mM ATP 15% Polyethylene glycol (PEG 6000) pH 7.6 @25°C
T4 RNA Ligase 1 (ssRNA Ligase)
NEB Storage Conditions: 10 mM Tris-HCl 50 mM KCl 1 mM Dithiothreitol 0.1 mM EDTA 50% Glycerol pH 7.5 @ 25°C 1X T4 RNA Ligase Reaction Buffer: 50 mM Tris-HCl 10 mM MgCl2 1 mM Dithiothreitol pH 7.5 @ 25°C
Shrimp Alkaline Phosphatase (SAP) (1 hour at 37C for 3' ends. 65C 15 mins inactivate)
Fermentas Storage Buffer 25 mM Tris-HCl (pH 7.6 at 4°C) 1 mM MgCl2 0.1 mM ZnCl2 and 50% (v/v) glycerol. 10X Reaction Buffer 0.1 M Tris-HCl (pH 7.5 at 37°C) 0.1 M MgCl2 1 mg/ml BSA.
Antarctic Phosphatase (37C for 30 mins. inactivate 5 min at 65C, inactivtes really easily)
NEB Storage Conditions: 10 mM Tris-HCl 1 mM MgCl2 0.01 mM ZnCl2 1 mM Dithiothreitol 50% Glycerol pH 7.4 @ 25°C 1X Antarctic Phosphatase Reaction Buffer: 50 mM Bis-Tris-Propane-HCl 1 mM MgCl2 0.1 mM ZnCl2 pH 6.0 @ 25°C
T4 Polynucleotide Kinase (PNK) (37C for 30 mins. 65°C for 20 minutes inactivate)(30min for 5’-overhangs or blunt ends, 60 min for 3’-overhangs.)
NEB 1X T4 Polynucleotide Kinase Reaction Buffer: 70 mM Tris-HCl 10 mM MgCl2 5 mM Dithiothreitol pH 7.6 @ 25°C Storage Conditions: 10 mM Tris-HCl 50 mM KCl 0.1 µM ATP 1 mM Dithiothreitol 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C enzymatics: Supplied in 10 mM Tris-HCl 50 mM KCl 0.1 µM ATP 1.0 mM DTT 0.1 mM EDTA 50% glycerol pH 7.4 @ 25°C 10X Polynucleotide Kinase Buffer (B9040) 700 mM Tris-HCl 100 mM MgCl2 50 mM DTT pH 7.6 @ 25°C
T4 DNA ligase
Reaction Conditions 1X T4 DNA Ligase Reaction Buffer Incubate at 16°C 1X T4 DNA Ligase Reaction Buffer: 50 mM Tris-HCl 10 mM MgCl2 1 mM ATP 10 mM DTT pH 7.5 @ 25°C Storage Temperature -20°C Storage Conditions 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C
DNA polymerase 1
Invitrogen reaction buffer: 50 mM Tris-HCl (pH 7.8) 5 mM MgCl2 10 mM 2-mercaptoethanol 10 μg/ml BSA 20 μM dATP, dCTP, dGTP, dNTPs 1X NEBuffer 2: 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 1 mM DTT pH 7.9 @ 25°C Thermo scientific 10X Reaction Buffer 500 mM Tris-HCl (pH 7.5 at 25°C) 100 mM MgCl2 10 mM DTT.