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Buffer Preparation (Shi Lab) : 1. 1 M Tris-Hcl Buffers

The document provides instructions for preparing various buffers and solutions for use in molecular biology techniques like DNA electrophoresis, protein electrophoresis, immunoprecipitation, and chromatin immunoprecipitation. It includes recipes for Tris-based buffers, EDTA, TAE buffer, SDS-PAGE gel solutions, protein loading buffer, DNA loading buffer, transfer buffer, TBS buffer, lysis buffers, ChIP buffers, wash buffers, and elution buffer. Concentrations and pH values are specified to prepare buffers for techniques like gel electrophoresis, western blotting, and chromatin immunoprecipitation.

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Estari Mamidala
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141 views6 pages

Buffer Preparation (Shi Lab) : 1. 1 M Tris-Hcl Buffers

The document provides instructions for preparing various buffers and solutions for use in molecular biology techniques like DNA electrophoresis, protein electrophoresis, immunoprecipitation, and chromatin immunoprecipitation. It includes recipes for Tris-based buffers, EDTA, TAE buffer, SDS-PAGE gel solutions, protein loading buffer, DNA loading buffer, transfer buffer, TBS buffer, lysis buffers, ChIP buffers, wash buffers, and elution buffer. Concentrations and pH values are specified to prepare buffers for techniques like gel electrophoresis, western blotting, and chromatin immunoprecipitation.

Uploaded by

Estari Mamidala
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Buffer Preparation (Shi Lab)

1. 1 M Tris-HCl Buffers
pH

Volume (L)

TrisBase (g)

HCl (ml)

pH 7.0

242.2

150-155

pH 7.5

242.2

120-125

pH 8.0

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,
0.4% SDS

363.3

50-60

80 ml

4x Upper gel buffer


0.5 M Tris-Cl, pH 6.8,
0.4% SDS

121.1

70-80

80 ml

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
250 mM TrisHCl pH6.8
10% SDS
30% Glycerol
5% -mercapitalethanol (or 0.5M DTT)
0.02% bromophenol blue

Stock solution
1M

1%

Add volume
25 ml
10 g
30 ml
5 ml
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 m
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 m 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 M (~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
5 mM PIPES pH 8.0
85 mM KCl
1% NP40
ddH2O

Stock
0.5 M
3M
10%

Vol for 500 ml


5 ml
14 ml
50 ml
433 ml

ChIP Nuclei Lysis buffer


50 mM Tris-Cl pH 8.0
10 mM EDTA
1% SDS
ddH2O

1M
0.5 M
10%

500 ml
25 ml
10 ml
50 ml
425 ml

ChIP Dilution buffer


16.7 mM Tris-Cl pH 8.0
167 mM NaCl
0.01% SDS
1.1% Trition X 100
1.2 mM EDTA
ddH2O

1M
4M
10%
10%
0.5 M

500 ml
8.4 ml
20.8 ml
0.5 ml
55 ml
1.2 ml
414 ml

ChIP Dialysis buffer


-Rabbit
50 mM Tris-Cl pH 8.0
0.2% Sarkosyl
2 mM EDTA
ddH2O
ChIP Dialysis buffer
-Mouse
50 mM Tris-Cl pH 8.0
2 mM EDTA
ddH2O
ChIP Wash buffer-Rabbit
100 mM Tris, pH 9.0
500 mM LiCl (MW 42.4)
1% NP40
1% Deoxycholic acid (sodium
salt. MW 414.5)
ChIP Wash buffer-mouse
100 mM Tris, pH 8.0,
500 mM LiCl (MW 42.4)
1% NP40
1% Deoxycholic acid (sodium
salt. MW 414.5)
ChIP Elution buffer
50 mM NaHCO3
1% SDS

1000 ml
1M
20%
0.5 M

50 ml
10 ml
4 ml
926 ml
1000 ml

1M
0.5 M

1M
10%

1M
10%

50 ml
4 ml
946 ml
1000 ml
100 ml
21.2 g
100 ml
10 g
1000 ml
100 ml
21.2 g
100 ml
10 g
Make fresh

1M
10%

1.25 M Glycine 200ml


(MW=75) 18.8 g
0.5M PIPES 200 ml
30.2g PIPES, 17-19 ml
10 M NaOH
1 M NaHCO3 (MW
84) 4.2g/50 ml
5 ml elution buffer:
0.5 ml 10% SDS
21 mg NaHCO3

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