Buffer Preparation (Shi 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)
4L
968 g Tris
228.4 ml glacial acetic acid
400 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, 2 363.3 50-60 80 ml
0.4% SDS
4x Upper gel buffer
0.5 M Tris-Cl, pH 6.8, 2 121.1 70-80 80 ml
0.4% SDS
4.1 10% SDS
2L:
200g SDS into 2 L, heat to 68oC for solubility. pH ~6.6.
5. 5X SDS Loading Sample Buffer
100 ml
Stock solution Add volume
250 mM TrisHCl pH6.8 1M 25 ml
10% SDS 10 g
30% Glycerol 30 ml
5% β-mercapitalethanol (or 0.5M DTT) 5 ml
0.02% bromophenol blue 1% 2 ml
6. 6X DNA loading sample buffer:
(40% sucrose, 0.01-0.02% BPB)
100 ml
Add 40 g sucrose to 50 ml ddH2O, add 2 ml 1% BPB solution, adjust to 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 SDS-PAGE running buffer
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,
~50-60 ml HCl
to pH8.0
9.1 TBS-T (1x)
20L
2L 10x TBS
200 ml 10% Tween20 (final 0.1% v/v)
ddH2O to 20 L
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 0.1M
250 ml: 3.4 g ZnCl.
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.
1.1. GST pull-dwon binding buffer (1 M)
(50 mM Tris 7.5, 300 mM NaCl, 0.05% NP-40.)
1L
50 ml 1M Tris HCl 7.5;
75 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% NP-40, 0.1% SDS)
1L
50 ml 1 M Tris 7.4,
37.5 ml 4 M NaCl,
4 ml 0.5 M EDTA,
10 ml NP-40.
10 ml 10% SDS.
3. Cell Lysis Buffer (Flag-IP buffer)
(50 mM TrisHCl pH7.4, 250 mM NaCl, 0.5% Triton X100, 10% glycerol, 1 mM
DTT, PMSF, PI (Roche))
1L
50 ml 1 M Tris 7.4,
62.5 ml 4 M NaCl,
5 ml Triton X-100,
1 ml 1 M DTT,
100 ml glycerol.
ChIP Solutions
ChIP Sweeling buffer Stock Vol for 500 ml
5 mM PIPES pH 8.0 0.5 M 5 ml
85 mM KCl 3M 14 ml
1% NP40 10% 50 ml
ddH2O 433 ml
ChIP Nuclei Lysis buffer 500 ml
50 mM Tris-Cl pH 8.0 1M 25 ml
10 mM EDTA 0.5 M 10 ml
1% SDS 10% 50 ml
ddH2O 425 ml
ChIP Dilution buffer 500 ml
16.7 mM Tris-Cl pH 8.0 1M 8.4 ml
167 mM NaCl 4M 20.8 ml
0.01% SDS 10% 0.5 ml
1.1% Trition X 100 10% 55 ml
1.2 mM EDTA 0.5 M 1.2 ml
ddH2O 414 ml
ChIP Dialysis buffer 1000 ml
-Rabbit 1.25 M Glycine 200ml
50 mM Tris-Cl pH 8.0 1M 50 ml (MW=75) 18.8 g
0.2% Sarkosyl 20% 10 ml
2 mM EDTA 0.5 M 4 ml 0.5M PIPES 200 ml
ddH2O 926 ml 30.2g PIPES, 17-19 ml
10 M NaOH
ChIP Dialysis buffer 1000 ml
-Mouse 1 M NaHCO3 (MW
50 mM Tris-Cl pH 8.0 1M 50 ml 84) 4.2g/50 ml
2 mM EDTA 0.5 M 4 ml
ddH2O 946 ml
5 ml elution buffer:
ChIP Wash buffer-Rabbit 1000 ml 0.5 ml 10% SDS
100 mM Tris, pH 9.0 1M 100 ml 21 mg NaHCO3
500 mM LiCl (MW 42.4) 21.2 g
1% NP40 10% 100 ml
1% Deoxycholic acid (sodium 10 g
salt. MW 414.5)
ChIP Wash buffer-mouse 1000 ml
100 mM Tris, pH 8.0, 1M 100 ml
500 mM LiCl (MW 42.4) 21.2 g
1% NP40 10% 100 ml
1% Deoxycholic acid (sodium 10 g
salt. MW 414.5)
ChIP Elution buffer Make fresh
50 mM NaHCO3 1M
1% SDS 10%
