Buffer Preparation (Gozani Lab)

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)

2 L

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

Add volume

250 mM TrisHCl pH6.8

2 M

12.5 ml

10% SDS

10 g

30% Glycerol

30 ml

5% β-mercapitalethanol (or 0.5M DTT)

5 ml

0.02% bromophenol blue

0.04%

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% 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.

4. H-Lysis solution:

(0.25M sucrose (MW=342), 3 mM CaCl2, 1 mM Tris pH8.0, 0.5% NP-40)

500 ml

43 g sucrose

1.5 ml 1 M Cacl2

0.5 ml 1 M Tris pH8.0

25 ml 10% NP-40

add ddH2O to 100 ml

Filter sterilize, store at 4oC.

5. H-Wash solution:

(300 mM NaCl, 5 mM MgCl2, 5 mM DTT, 0.5% NP-40)

500 ml

37.5 ml 4 M NaCl

2.5 ml MgCl2

2.5 ml DTT

25 ml 10% NP-40

6. H-Extraction solution (Histones Extraction):

(0.5 M HCl, 10% glycerol, 0.1 M 2-mercaptoethylamine-HCl (MW: 113.6)).

50 ml

2.25 ml HCl (11.2 M)

10 ml 50% glycerol.

0.55 g 2-mercaptoethylamine-HCl (Sigma name: cysteamine hydrochloride)

Recipe of making SDS-PAGE

SDS-PAGE

12% resolve gel

10% resolve gel

4% stacking gel 10 ml

4x buffer

10

5

2.5

40% Acr-Bis

6

5

1

ddH2O

4

10

6.5

10% APS (ul)

100

100

100

20 ml

(For 4x1mm plate)

TEMED (ul)

20

20

20

4x buffer

5

5

2.5

30% Acr-Bis

8

6.6

1.3

ddH2O

7

8.4

6.2

10% APS (ul)

100

100

100

20 ml

(For 4x1mm plate)

TEMED (ul)

20

20

20

Voltage

Time

Buffer

Volume

Electrophoresis

150V/200V (15/30 mA)

1 h

Tris/Glycine/SDS

300 ml tank

Semi-Dry Transfer

5V

(40-60 mA/gel)

1-2 h

20% Methanol

1x SDS running buffer

Make 500 ml for 4 gels

Agarose

100V

30min-1h

10 μl EB to 100 ml agarose/TAE

350 ml tank