Buffer Preparation

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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. Autoclavable.
11. NaOH 10 M
0.5 L: 200 g
12. NaAc 3 M
500 ml: add 204 g NaAc.3H2O (FW 136), adjust pH by glacial acetic acid (~60 ml) to pH5.2. Autoclavable.
13. MgCl2 1M
500 ml: Add 101.65 g MgCl2.6H2O into 500 ml ddH2O. Autoclavable. 14. CaCl2 1M
400 ml: Add 58.8 g CaCl2.2H2O (FW 147), filter for sterilization. Dilute 10x to make 100 mM CaCl2.
15. MgSO4 1M
500 ml: Add 123.3 g MgSO4.7H2O into 500 ml ddH2O. Autoclavable.

16. ZnCl 0.5M
50 ml: 3.4 g ZnCl to 50 ml.

Stock in -20oC

1. IPTG (1 M)
1 g IPTG (FW 238.3) resolved in 4.2 ml (~4 ml) ddH2O, filter through 0.22 µm filters, aliquot 1 ml in eppendorf. Store at -20oC.
2. DTT (1 M)
5 g DTT (FW 154.25) resolved in 32.5 ml (~30 ml)10 mM NaAc (pH 5.2), filter through 0.22 µm filters, aliquot 1 ml in eppendorf. Store at -20oC. 3. X-gal (20mg/ml)
Add 5 ml (~4.8 ml) DMSO into 100 mg X-gal bottom (FW 408.24). Store at -20oC. 4. PMSF (100 mM, =17.4 mg/ml)
Resolve 1.74g PMSF (MW 174) in isoproponal, total 100 ml. Aliquot and store at 20oC or R.T.. 5. Carbencillin or Ampcillin (50 mg/ml) in water. 1000x
2.5 g 50 ml.
6. Kanamycin (10 mg/ml) in water. 200x
0.5 g 50 ml.
7. Chloramphenicol (34 mg/ml) in ethanol. 200x
1.7 g/ 50 m l.
8. lysozyme 50 mg/ml, 1000x.
2.5 g/ 50 ml.
9. TSA (MW 303):
Add 1.32 ml Ethanol into each vial (1 mg?) to make the TSA stock 2.5 mM, 5000x. Final concentration of TSA in the cell culture is 0.5 μM (~150 ng/ml).

Solutions.
1. Bacteria lysis buffer (GST pull-dwon binding buffer)
(50 mM Tris 7.5, 150 mM NaCl, 0.05% NP-40.)
1L
50 ml 1M Tris HCl 7.5;
37.5 ml 4 M NaCl;
5 ml 10% NP-40.
ddH2O to 1L.
1.2. GST pull-dwon binding buffer (1 M)
(50 mM Tris 7.5, 1 M NaCl, 1% NP-40.)
500 ml
25 ml 1M Tris HCl 7.5;
125 ml 4 M NaCl;
50 ml 10% NP-40.
ddH2O to 500ml.
2. RIPA Buffer
(50 mM TrisHCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1%...
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