Buffer Preparation
Buffer Preparation (Gozani Lab)
1. 1 M Tris-HCl Buffers pH Volume (L)
TrisBase (g)
HCl (ml)
pH 7.0
2
242.2
150-155
pH 7.5
2
242.2
120-125
pH 8.0
2
242.2
80-85
Autoclavable.
2. EDTA 0.5 M (pH8.0)
0.5M, 1L: 148 g EDTA
+ ~30-40 g NaOH to adjust pH
(or 186 g EDTA-Na.2H2O + ~20 g NaOH)
Note: pH adjusted by NaOH is essential for solubility. Autoclavable.
3. TAE DNA Electrophoresis Buffer (50 X)
(2 M Tris, 50 mM EDTA)
2L
484 g Tris
114.2 ml glacial acetic acid
200 ml 0.5 M EDTA 8.0
To make 1x TAE 20 L, add 400 ml 50X buffer into 19.6 L ddH2O.
4. SDS-PAGE Gel Solutions
Vol (L)
Tris (g)
HCl (ml)
10% SDS (ml)
4x Lower gel buffer
1.5 M Tris-Cl, pH 8.8,
0.4% SDS
2
363.3
50-60
80 ml
4x Upper gel buffer
0.5 M Tris-Cl, pH 6.8,
0.4% SDS
2
121.1
70-80
80 ml
4.1 10% SDS
1L:
100g SDS into 1 L, heat to 68oC for solubility. pH ~6.6.
5. 5X SDS Loading Sample Buffer
100 ml
Stock solution
250 mM TrisHCl pH6.8
2M
10% SDS
30% Glycerol
5% β-mercapitalethanol (or 0.5M DTT)
0.02% bromophenol blue
0.04%
Add volume
12.5 ml
10 g
30 ml
5 ml
52 ml
6. 6X DNA loading sample buffer:
(40% sucrose, 0.01-0.02% BPB)
100 ml
Add 40 g sucrose to 50 ml 0.04% BPB solution, adjust final volume 100 ml.
7. SDS-PAGE Electrophoresis Running Buffer (10x)
(1x: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH8.3)
10 L.
303 g Trisbase (FW 121.1)
1440 g glycine (FW 75.07)
100 g SDS
No need to adjust pH
8. Transfer Buffer without SDS (10x)
(1x: 25 mM Tris, 192 mM glycine, pH8.3)
10 L
303 g Trisbase,
1440 g glycine
No need to adjust pH
8.1 Transfer Buffer (1x)
500 ml
50 ml of 10x Transfer buffer (without SDS) or 10x SDS-PAGE running buffer (w/ SDS)
100 ml of Methanol (final 20% methanol)
350 ml ddH2O
9. TBS (10x)
(1x: 150 mM NaCl, 10 mM Tris pH8.0)
10 L
876.6 g NaCl (FW 58.44),
121.1 g Tris,
~40 ml HCl to pH8.0
9.1 TBS-T (1x)
1L
100 ml 10x TBS
10 ml 10% Tween20 (final 0.1% v/v)
890 ml ddH2O
9.2 Block buffer
(5% Nonfat milk in TBS-T)
5g milk in 100 ml TBST
10. NaCl 4 M
2 L: 467.5 g NaCl.
1. 1 M Tris-HCl Buffers pH Volume (L)
TrisBase (g)
HCl (ml)
pH 7.0
2
242.2
150-155
pH 7.5
2
242.2
120-125
pH 8.0
2
242.2
80-85
Autoclavable.
2. EDTA 0.5 M (pH8.0)
0.5M, 1L: 148 g EDTA
+ ~30-40 g NaOH to adjust pH
(or 186 g EDTA-Na.2H2O + ~20 g NaOH)
Note: pH adjusted by NaOH is essential for solubility. Autoclavable.
3. TAE DNA Electrophoresis Buffer (50 X)
(2 M Tris, 50 mM EDTA)
2L
484 g Tris
114.2 ml glacial acetic acid
200 ml 0.5 M EDTA 8.0
To make 1x TAE 20 L, add 400 ml 50X buffer into 19.6 L ddH2O.
4. SDS-PAGE Gel Solutions
Vol (L)
Tris (g)
HCl (ml)
10% SDS (ml)
4x Lower gel buffer
1.5 M Tris-Cl, pH 8.8,
0.4% SDS
2
363.3
50-60
80 ml
4x Upper gel buffer
0.5 M Tris-Cl, pH 6.8,
0.4% SDS
2
121.1
70-80
80 ml
4.1 10% SDS
1L:
100g SDS into 1 L, heat to 68oC for solubility. pH ~6.6.
5. 5X SDS Loading Sample Buffer
100 ml
Stock solution
250 mM TrisHCl pH6.8
2M
10% SDS
30% Glycerol
5% β-mercapitalethanol (or 0.5M DTT)
0.02% bromophenol blue
0.04%
Add volume
12.5 ml
10 g
30 ml
5 ml
52 ml
6. 6X DNA loading sample buffer:
(40% sucrose, 0.01-0.02% BPB)
100 ml
Add 40 g sucrose to 50 ml 0.04% BPB solution, adjust final volume 100 ml.
7. SDS-PAGE Electrophoresis Running Buffer (10x)
(1x: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH8.3)
10 L.
303 g Trisbase (FW 121.1)
1440 g glycine (FW 75.07)
100 g SDS
No need to adjust pH
8. Transfer Buffer without SDS (10x)
(1x: 25 mM Tris, 192 mM glycine, pH8.3)
10 L
303 g Trisbase,
1440 g glycine
No need to adjust pH
8.1 Transfer Buffer (1x)
500 ml
50 ml of 10x Transfer buffer (without SDS) or 10x SDS-PAGE running buffer (w/ SDS)
100 ml of Methanol (final 20% methanol)
350 ml ddH2O
9. TBS (10x)
(1x: 150 mM NaCl, 10 mM Tris pH8.0)
10 L
876.6 g NaCl (FW 58.44),
121.1 g Tris,
~40 ml HCl to pH8.0
9.1 TBS-T (1x)
1L
100 ml 10x TBS
10 ml 10% Tween20 (final 0.1% v/v)
890 ml ddH2O
9.2 Block buffer
(5% Nonfat milk in TBS-T)
5g milk in 100 ml TBST
10. NaCl 4 M
2 L: 467.5 g NaCl.