Brandon:Protocols/enzyme rxn buffers

From ZhangLabWiki
Jump to navigation Jump to search

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.