Organic substances.
pKa
and temperature dependence of pH for common buffers.
ATP
0.1M
Betaine
5M
Cresol red (Na)
50mM
DTT
1M, 2.2M
dNTP’s
100mM
EDTA
0.5M
EtBr
10mg/ml
Gelatin
2%
Glucose
1; 1.5; 2M
Guanidine HCl
1-8M
HEPES
1M
Imidazol
2M
Paraformaldehyde
37%
PEG
40%
PMSF
100mM
Retinoic acid
10mM
Sucrose
1; 2; 2.5M
Tris Cl
1M
Temperature dependence of pH for TrisCl.
Tricine
1M
Triethanolamine
1M
Urea
1-10M
Acids and alkalis.
Summary table.
NaOH
10M, 1M
KOH
5M
TCA
100%
Detergents.
N-Lauroylsarcosine Na
10%
SDS
10%
Organic solvents.
Phenol
EthanolEtOH
Preparation of 100% EtOH.
Supplement.
|
Densities of some solutions are available on the page "
Densities of acids, alkali and organic substances
".
See also:
- Buffer design
- online calculations of large list of buffers (Prof. R.Beynon) on the page
Java based Molecular Biologist's Workbench
EMBL.
- Buffer Calculator
on the site of LabVelocity. Registration is necessary (free).
<center><font>About the recalculation of recipes for the arbitrary volumes:<br />
<br />
</font>
</center>
|
|
Organic substances.
pKa
and temperature dependence of pH for common buffers.
Buffer
|
Mw
|
pKa
20o
C
|
Working
range
|
delta pKa
per 10o
C
|
MES
2-(N
-morpholino)ethanesulfonic acid
|
195.2
|
6.15
|
5.5-6.7
|
-0.110
|
Bis-Tris
bis
(2-hydroxyethyl)iminotris
(hydroxymethyl)methane
|
209.2
|
6.5
|
5.8-7.2
|
|
ADA
N
-(2-acetamido)-2-imidoacetic acid
|
190.2
|
6.60
|
6.0-7.2
|
-0.110
|
PIPES
piperasine-N,N'-bis
(2-ethanesulfonic acid)
|
302.4
|
6.80
|
6.1-7.5
|
-0.085
|
ACES
2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid
|
182.2
|
6.90
|
6.1-7.5
|
-0.200
|
MOPSO
3-(N
-morpholino)-2-hydroxypropanesulfonic acid
|
225.3
|
6.9
|
6.2-7.6
|
|
Imidazol - HCl
|
68.08
|
6.95
|
6.2-7.8
|
|
Bis-Tris
Propane
1,3-bis
[tris
(hydroxymethyl)methylamino]propane
|
282.3
|
6.undefined
|
6.3-9.5
|
|
BES
N,N-bis
(2-hydroxyethyl)-2-aminoethanesulfonic acid
|
213.2
|
7.15
|
6.4-7.8
|
-0.160
|
MOPS
3-(N
-morpholino)propanesulfonic acid
|
209.3
|
7.20
|
6.5-7.9
|
-0.013
|
TES
N-tris
(hydroxymethyl)methyl-2-aminoethanesulfonic acid
|
229.2
|
7.50
|
6.8-8.2
|
-0.200
|
HEPES
N
-(2-hydroxyethyl)piperazine-N'
-(2-ethanesulfonic acid)
|
238.3
|
7.55
|
6.8-8.2
|
-0.140
|
DIPSO
3-[N,N-bis
(2-hydroxyethyl)amino]-2-hydroxyprpanesulfonic acid
|
243.3
|
7.6
|
7.0-8.2
|
|
TAPSO
3-[N-tris
(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid
|
259.3
|
7.6
|
7.0-8.2
|
|
HEPPSO
N
-(2-hydroxyethyl)piperazine-N'
-(2-hydroxypropanesulfonic acid)
|
268.3
|
7.8
|
7.1-8.5
|
|
POPSO
piperazine-N,N'-bis
(2-hydroxypropanesulfonic acid)
|
362.4
|
7.8
|
7.2-8.5
|
|
TEA
triethanolamine
|
149.2
|
7.8
|
7.3-8.3
|
|
EPPS
N
-(2-hydroxyethyl)piperazine-N'
-(3-propanesulfonic acid)
|
252.3
|
8.0
|
7.3-8.7
|
|
Tricine
N-tris
(hydroxymethyl)methylglycine
|
179.2
|
8.15
|
7.4-8.8
|
-0.210
|
Tris (TRIZMA)
tris
(hydroxymethyl)aminomethane
|
121.1
|
8.30
|
7.0-9.1
|
-0.310
|
Bicine
N,N-bis
(2-hydroxyethyl)glycine
|
163.2
|
8.35
|
7.6-9.0
|
-0.180
|
TAPS
N-tris
(hydroxymethyl)methyl-3-aminopropanesulfonic acid
|
243.3
|
8.4
|
7.7-9.1
|
|
Glycylglycine
|
|
8.40
|
|
-0.280
|
AMPSO
3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid
|
227.3
|
9.0
|
8.3-9.7
|
|
CHES
1-(N
-cyclohexylamino)ethanesulfonic acid
|
207.3
|
9.3
|
8.6-10.0
|
|
CAPSO
3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid
|
237.3
|
9.6
|
8.9-10.3
|
|
AMP
2-amino-2-methyl-1-propanol
|
89.1
|
9.7
|
9.0-10.5
|
|
CAPS
3-(cyclohexylamino)-1-propanesulfonic acid
|
221.3
|
10.4
|
9.7-11.1
|
|
|
ATP
C10
H14
N5
O13
P3
Na2
Mw=551.1 g/M; (store at -20o
C).
|
Conc.
|
Stock
|
1ml
|
|
ATP
|
0.1M
|
551.1g/M
|
55.1mg
|
|
H2
O
|
|
mQ
|
|
|
|
0.1M ATP (pH 7.2): 5.71% (w/w) ATP, 84.86% (w/w) H2
O, 9.43% (w/w) 2M Tris base.
- adjust to pH7.5 by 2M NaOH (~70-80µl);
- prepare about 700µl, dilute 2000 times (it is the final dilution in the spectrofotometric cell), check the optical density:
C[M]=A259
/15.4x103
=A259 1:2000
x0.130
- adjust the final volume.
Betaine
monohydrate C5
H11
NO2
xH2
O; Mw=135.2g/M; (store at 4o
C).
|
Conc.
|
Stock
|
%(w/w)
|
100ml
|
200ml
|
700ml
|
|
Betaine
|
135.2g/M
|
5M
|
63.00
|
67.6g
|
135.2g
|
473.2g
|
|
H2
O
|
|
mQ
|
37.00
|
39.74ml
|
79.49ml
|
278.2ml
|
|
|
p~1.073g/ml
Cresol red (Na)
50mM (store at +4, -20o
C):
|
Conc.
|
Stock
|
1ml
|
50ml
|
|
Cresol red (Na)
|
50mM
|
404.4g/M
|
20.2mg
|
1.01g
|
|
H2
O
|
|
mQ
|
|
|
|
|
- Cresol Red (Na salt) is a very convenient dye. At a concentration of about 0.2mM it is compatible with restriction digestion, PCR, sequencing. It may be used as marker for electrophoresis;
- the color of the dye is pH-dependent (red, if pH>7.5; yellow, if pH <7.0). It is possible to use Cresol Red as pH-indicator (i) for denaturation of double-stranded DNA in sequencing, (ii) for silica-purification of DNA from agarose gel.
DTT
C4
H10
O2
S2
Mw=154.2g/M; (store at -20o
C).
|
Conc.
|
Stock
|
%(w/w)
|
1ml
|
5ml
|
10ml
|
20ml
|
|
DTT
|
154.2g/M
|
1M
|
14.55
|
0.155g
|
0.773g
|
1.55g
|
3.09g
|
|
AcNa, pH 5.2
|
|
10mM
|
85.45
|
905 µl
|
4.53ml
|
9.05ml
|
18ml
|
|
|
p~1.06g/ml
|
Conc.
|
Stock
|
1ml
|
5ml
|
10ml
|
20ml
|
|
DTT
|
154.2g/M
|
2.2M
|
0.339g
|
1.696g
|
3.394g
|
6.787g
|
|
AcNa, pH 5.2
|
|
10mM
|
|
|
|
|
|
|
- filter sterililize;
- no DEPC treatment.
Preparation of dNTP's.
The quick protocol for ~100mM stock.
- dilute all four dNTP's (250mg of each) in 3.676ml of H2
O;
- + 424µl 5M NaOH;
- check the pH: ~0.5µl on pH-paper.
Accurate protocol for 100mM stocks preparation.
- add the necessary quantity (see table) of H2
O and Tris base 1M (it is possible to take the volume of salt as ~150µl);
|
|
Mw
|
V(H2O)
|
V(Tris base 1M)
|
V(final)
|
dATP
|
C10
H14
N5
O12
P3
Na2
x3H2
O
|
589.2
|
3.24ml
|
850 µl
|
4.24ml
|
dGTP
|
C10
H13
N5
O13
P3
Na3
x2H2
O
|
609.2
|
3.55ml
|
400 µl
|
4.10ml
|
dCTP
|
C9
H13
N3
O13
P3
Na3
x2H2
O
|
569.1
|
3.44ml
|
800 µl
|
4.39ml
|
dTTP
|
C10
H14
N2
O14
P3
Na3
x2H2
O
|
584.1
|
3.73ml
|
400 µl
|
4.28ml
|
|
- check the pH: ~0.5µl on pH-paper;
- check the quality and concentration (it is useful to take final dilution 1:5000 (~20µM). In this case the optical density will be in the region of Am
~0.3 - the most accurate range for spectrophotometer).
Concentration is c[mM]=k1:5000
xAm
.
Quality:
dATP
pH 7.0
|
A250
/A260
=0.80+
0.03
A280
/A260
=0.12+
0.02
|
dCTP
pH 2.0(!)
|
A250
/A260
=0.45+
0.03
A280
/A260
=2.10+
0.15
A290
/A260
=1.60+
0.10
|
dGTP
pH 7.5
|
A250
/A260
=1.18+
0.04
A280
/A260
=0.67+
0.03
A290
/A260
=0.28+
0.03
|
dTTP
pH 7.5
|
A250
/A260
=0.65+
0.03
A280
/A260
=0.73+
0.03
|
|
Concentration:
|
Mw
|
Am
|
pH
|
E
|
k (for 1:5000)
|
dATP[Na2
]
|
589.2
|
259
|
7.0
|
15.2x103
|
328.9
|
dGTP[Na3
]
|
609.2
|
253
|
7.5
|
13.7x103
|
365.0
|
dCTP[Na3
]
|
569.1
|
280
|
2.0 (!!!)
|
13.0x103
|
384.6
|
dTTP[Na3
]
|
584.1
|
267
|
7.5
|
9.6x103
|
520.8
|
|
|
|
|
|
|
ATP [Na4
]
|
595.1
|
259
|
|
15.4x103
|
|
CTP [Na4
]
|
571.1
|
280
|
|
13.0x103
|
|
GTP [Na4
]
|
611.1
|
252
|
|
13.7x103
|
|
UTP [Na4
]
|
572.1
|
262
|
|
10.2x103
|
|
|
Concentration: c[M]=Amax
/E; Amax
= maximum of absorption.
EDTA
C10
H14
O8
N2
Na2
x2H2
O; Mw=372.3g/M; pH = 8.0 (store at 4o
C).
|
Conc.
|
Stock
|
%(w/w)
|
50 ml
|
100 ml
|
150 ml
|
250 ml
|
|
EDTA
|
0.5 M
|
372.3g/M
|
16.98
|
9.31g
|
18.62g
|
27.92g
|
46.55g
|
|
NaOH
|
~0.5 M
|
40g/M
|
1.82
|
1.014g
|
2.028g
|
3.042g
|
5.07g
|
|
H2
O
|
|
mQ
|
81.19
|
44.48ml
|
88.95ml
|
133.4ml
|
222.4ml
|
|
|
|
Conc.
|
Stock
|
10 ml
|
50 ml
|
100 ml
|
150 ml
|
250 ml
|
|
EDTA
|
0.5 M
|
372.3g/M
|
1.86g
|
9.31g
|
18.62g
|
27.92g
|
46.55g
|
|
NaOH
|
~0.5 M
|
10M
|
507µl
0.674g
|
2.535ml
3.37g
|
5.07ml
6.74g
|
7.61ml
10.11g
|
12.68ml
16.85g
|
|
H2
O
|
|
mQ
|
8.42ml
|
42.12ml
|
84.24ml
|
126.4ml
|
210.6ml
|
|
|
p~1.096g/ml
- EDTA is not soluble at acidic pH; it is necessary to add alkali gradually and to control pH;
- do not treat by DEPC.
EtBr
Ethidium bromide, C21
H20
N3
Br; Mw=394.3g/M; (store at NT in the dark);
|
Conc.
|
5ml
|
10ml
|
50ml
|
|
EtBr
|
10mg/ml
|
50mg
|
100mg
|
500mg
|
|
|
- soluble in H2
O, EtOH, chloroform;
- concerning carcinogenic properties of EtBr. The only data that we found in the literature is that in mutagenic test (on bacteria) 90µg of EtBr gave the same results as the smoke concentrate from one cigarette.
Gelatin
(store at 4o
C).
|
Conc.
|
Stock
|
10ml
|
50ml
|
100ml
|
|
Gelatin
|
2%
|
solid
|
0.2g
|
1.0g
|
2.0g
|
|
H2
O
|
|
mQ
|
|
|
|
|
|
- sterilize by autoclaving.
Glucose
C6
H12
O6
xH2
O, Mw=198.17g/M (store at 4o
C)
|
Conc.
|
Stock
|
%(w/w)
|
50ml
|
100ml
|
250ml
|
|
Glucose
|
2M
|
198.17g/M
|
34.972
|
19.82g
|
39.63g
|
99.09g
|
|
H2
O
|
|
mQ
|
65.028
|
36.85ml
|
73.70ml
|
184.24ml
|
|
|
|
1000ml/
|
198.17g/M
|
1M
|
1.5M
|
2M
|
Glucose [g]
|
198.17
|
297.255
|
396.34
|
H2
O [ml]
|
868.23
|
802.745
|
736.96
|
%(w/w)
|
18.583
|
27.023
|
34.972
|
H2
O %(w/w)
|
81.417
|
72.977
|
65.028
|
p (g/ml) 20o
C
|
1.0664
|
1.1
|
1.1333
|
|
|
Guanidine HCl (GuHCl)
CH5
N3
xHCl, Mw=95.53g/M
|
Molarity; 1000ml /
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
GuHCl
|
95.53g
|
191.06g
|
286.59g
|
382.12g
|
477.65g
|
573.18g
|
668.71g
|
764.24g
|
H2
O (mQ)
|
924.2ml
|
854ml
|
783.7ml
|
711.7ml
|
639.8ml
|
567.2ml
|
494.3ml
|
420.7ml
|
GuHCl %(w/w)
|
9.37
|
18.28
|
26.78
|
34.93
|
42.75
|
50.26
|
57.50
|
64.50
|
H2
O %(w/w)
|
90.63
|
81.72
|
73.22
|
65.07
|
57.25
|
49.74
|
42.50
|
35.50
|
p (g/ml)
|
1.020
|
1.045
|
1.070
|
1.094
|
1.117
|
1.140
|
1.163
|
1.185
|
|
- solubility: at 25o
C - 8.54M, 5o
C - >
8M;
- A260
(6M in H2
O)<0.03;
- it is possible to take the "partial density of GuHCl" as 0.763 in calculations of solutions.
|
HEPES
|
Conc.
|
Stock
|
%(w/w)
|
1L
|
|
C8
H18
N2
O4
S
|
1M
|
238.3g/M
|
22.40
|
238.3g
|
|
H2
O
|
|
mQ
|
77.60
|
825.7ml
|
|
|
p=1.064
HEPES, 1M, 1L
Desired pH
|
KOH, 5M
[1000ml]
|
|
|
5.25
|
0ml
|
0ml
|
5.35
|
0.5ml
|
|
5.75
|
3.5ml
|
|
6.03
|
7ml
|
|
6.24
|
12ml
|
|
6.59
|
22ml
|
|
6.71
|
32ml
|
|
6.88
|
45ml
|
|
7.00
|
50ml
|
|
7.10
|
60ml
|
|
7.25
|
80ml
|
|
7.37
|
92.5ml
|
|
|
Imidazol
C3
H4
N2
, (store at 4o
C):
|
Conc.
|
Stock
|
50ml
|
100ml
|
|
Imidazole
|
2M
|
68.1g/M
|
6.81g
|
13.62g
|
|
H2
O
|
|
mQ
|
|
|
|
|
Paraformaldehyde
PFA (paraformaldehyde) 37% (for histochemistry it should be freshly prepared).
1. mix in the screw-cap tube:
PFA (solid) = 0.37g,
H2
O = 1.0ml
NaOH (1N) = 14µl;
2. solubilize in the boiling water bath (to heat ~1-3'; until pH will drop to ~7.0).
PEG
H(OCH2
CH2
)n
OH; (store at 4o
C).
|
Conc.
|
%(w/w)
|
10ml
|
50ml
|
100ml
|
150ml
|
200ml
|
|
PEG6000
|
40%
|
37.21
|
4.0g
|
20g
|
40g
|
60g
|
80g
|
|
H2
O
|
mQ
|
62.79
|
6.75g
|
33.75g
|
67.5g
|
101.25g
|
135.0g
|
|
|
p=1.075.
PMSF
(store at -20o
C)
|
Conc.
|
Stock
|
20ml
|
|
C7
H7
FO2
S
|
100mM
|
174.2g/M
|
0.348g
|
|
Isopropanol
|
|
|
20ml
|
|
|
Retinoic acid
all trans-Retinoic acid, Tretinoin, light-sensitive, (store at -20o
C):
|
Conc.
|
Stock
|
16.6ml
|
|
C20
H28
O2
|
10mM
|
300.4g/M
|
50mg
|
|
EtOH
|
|
>
96%
|
16.6ml
|
|
|
- stock solution is 10mM, working solution is freshly prepared 1mM in EtOH (it would be better to add pure EtOH to the control cells).
Sucrose
C12
H22
O11
, Mw=342.30g/M; 20o
C.
Densities and refraction indexes
of sucrose solutions.
|
Conc.
|
Stock
|
%(w/w)
|
50ml
|
100ml
|
250ml
|
|
Sucrose
|
1M
|
342.30g/M
|
30.330
|
17.115g
|
34.23g
|
85.58g
|
|
H2
O
|
|
mQ
|
69.670
|
39.315ml
|
78.63ml
|
196.58ml
|
|
|
|
1000ml/
|
342.30g/M
|
1M
|
2M
|
2.5M
|
Sucrose [g]
|
342.3
|
684.6
|
855.75
|
H2
O [ml]
|
786.3
|
570.4
|
460.35
|
%(w/w)
|
30.330
|
54.550
|
65.022
|
H2
O %(w/w)
|
69.670
|
45.450
|
34.978
|
p (g/ml) 18o
C
|
1.1286
|
1.2550
|
1.3161
|
|
Tris Cl
C4
H11
O3
N; Mw=121.1g/M; (store at 4o
C).
|
Conc.
|
Stock
|
50 ml
|
100 ml
|
150ml
|
200ml
|
|
Tris-base
|
1M
|
121.1g/M
|
6.06
|
12.11g
|
18.17g
|
24.22g
|
|
H2
O to the final weight
|
|
mQ
|
52.03g
|
104.06g
|
156.09g
|
208.12g
|
|
|
1M: p=1.0406
2M Tris base: 22.90%(w/w) Tris base, 77.10%(w/w) H2
O; p=1.058
- do not treat by DEPC;
- sterilize by autoclaving;
- pH of Tris-buffers is dependent from concentration. If to take 50mM solution as the original:
pH(500mM) => + 0.05
pH(5mM) => - 0.05
- pH drops on 0.028 when the temperature rise on 1o
C.
Temperature dependence of pH for Tris Cl 50mM.
pH at
|
g/50ml 1M or
g/liter for 0.05 M
|
|
5o
C
|
25o
C
|
37o
C
|
Tris HCl
|
Tris Base
|
H2
O
|
Tris HCl
|
Tris Base
|
H2
O
|
7.55
|
7.00
|
6.70
|
7.28
|
0.47
|
44.28
|
|
|
|
7.66
|
7.10
|
6.80
|
7.13
|
0.57
|
44.33
|
|
|
|
7.76
|
7.20
|
6.91
|
7.02
|
0.67
|
44.34
|
|
|
|
7.89
|
7.30
|
7.02
|
6.85
|
0.80
|
44.38
|
|
|
|
7.97
|
7.40
|
7.12
|
6.61
|
0.97
|
44.45
|
|
|
|
8.07
|
7.50
|
7.22
|
6.35
|
1.18
|
44.50
|
|
|
|
8.18
|
7.60
|
7.30
|
6.06
|
1.39
|
44.58
|
|
|
|
8.26
|
7.70
|
7.40
|
5.72
|
1.66
|
44.65
|
|
|
|
8.37
|
7.80
|
7.52
|
5.32
|
1.97
|
44.74
|
|
|
|
8.48
|
7.90
|
7.62
|
4.88
|
2.30
|
44.85
|
|
|
|
8.58
|
8.00
|
7.71
|
4.44
|
2.65
|
44.94
|
|
|
|
8.68
|
8.10
|
7.80
|
4.02
|
2.97
|
45.04
|
|
|
|
8.78
|
8.20
|
7.91
|
3.54
|
3.34
|
45.15
|
|
|
|
8.88
|
8.30
|
8.01
|
3.07
|
3.70
|
45.26
|
|
|
|
8.98
|
8.40
|
8.10
|
2.64
|
4.03
|
45.36
|
|
|
|
9.09
|
8.50
|
8.22
|
2.21
|
4.36
|
45.46
|
|
|
|
9.18
|
8.60
|
8.31
|
1.83
|
4.65
|
45.55
|
|
|
|
9.28
|
8.70
|
8.42
|
1.50
|
4.90
|
45.63
|
|
|
|
9.36
|
8.80
|
8.51
|
1.23
|
5.13
|
45.67
|
|
|
|
9.47
|
8.90
|
8.62
|
0.96
|
5.32
|
45.75
|
|
|
|
9.56
|
9.00
|
8.70
|
0.76
|
5.47
|
45.80
|
|
|
|
9.67
|
9.10
|
8.79
|
0.69
|
5.53
|
45.81
|
|
|
|
|
TrisCl: 250ml 1M
V HCl
|
V HCl
|
pH
|
|
0
|
10.44
|
|
1
|
9.54
|
|
2
|
9.26
|
|
3
|
9.08
|
|
3.7
|
8.98
|
5ml
|
|
8.76
|
6ml
|
|
8.67
|
|
10
|
8.4
|
11ml
|
|
8.22
|
|
14
|
8.13
|
13.5ml
|
|
8.06
|
14ml
|
|
8.04
|
15ml
|
|
7.97
|
|
17
|
7.92
|
16.5ml
|
|
7.82
|
16.7ml
|
|
7.8
|
17ml
|
|
7.77
|
18.3ml
|
|
7.63
|
18.6ml
|
|
7.59
|
|
20
|
7.75
|
|
23
|
7.4
|
|
Tricine
C6
H13
NO5
, Mw=179.2g/M;(store at 4o
C).
|
Conc.
|
Stock
|
50ml
|
|
Tricine
|
1M
|
179.2g/M
|
8.96g
|
|
H2
O
|
|
mQ
|
|
|
|
For 50ml:
Desired pH
|
5N KOH
[50ml]
|
|
8.30
|
7.0ml
|
|
8.38
|
7.5ml
|
|
8.47
|
8.0ml
|
|
8.5
|
8.15ml
|
|
8.6
|
8.5ml
|
|
8.68
|
9.0ml
|
|
8.78
|
9.5ml
|
|
8.90
|
10.0ml
|
|
|
Triethanolamine
1M (store at 4o
C):
|
Conc.
|
Stock
|
%(w/w)
|
50ml
|
|
Triethanolamine
|
1M
|
149.19g/M
|
14.69
|
7.46g
6.66ml
|
|
H2
O
|
|
mQ
|
85.31
|
43.34ml
|
|
|
|
Urea
CH4
N2
O, Mw=60.06g/M;
|
Molarity; 1000ml /
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
CH4
N2
O
[g]
|
60.06
|
120.12
|
180.18
|
240.24
|
300.30
|
360.36
|
420.42
|
480.48
|
540.54
|
600.60
|
H2
O
[ml]
|
950.6
|
905.8
|
861.3
|
817.0
|
771.6
|
726.7
|
681.2
|
635.7
|
590.0
|
544.1
|
p (g/ml)
|
1.011
|
1.026
|
1.041
|
1.057
|
1.072
|
1.087
|
1.102
|
1.116
|
1.131
|
1.145
|
CH4
N2
O
%(w/w)
|
5.94
|
11.71
|
17.30
|
22.72
|
28.02
|
33.15
|
38.16
|
43.05
|
47.81
|
52.47
|
H2
O %(w/w)
|
94.06
|
88.29
|
82.70
|
77.28
|
71.98
|
66.85
|
61.84
|
59.95
|
52.19
|
47.53
|
|
- solubility: at 25o
C: 10.49M, 5o
C: ~8M;
- A260
(6M in H2
O)<0.06;
- it is possible to take the "partial density of urea" as 0.763 in calculation of solutions.
|
Acids and alkalis.
Name:
|
Formula:
|
Mw
|
% (w/w)
|
[M]
|
g in 1L
of subst.
|
p [g/ml]
|
ml/L for
1M sol.
|
Sodium hydroxide
|
NaOH
|
40
|
50%
|
19.1
|
763
|
1.53
|
52.4
|
30.1%
|
10.0
|
400
|
1.329
|
100
|
10%
|
2.75
|
111
|
1.11
|
363.6
|
Potassium hydroxide
|
KOH
|
56.1
|
50%
|
13.5
|
757
|
1.52
|
74.1
|
23.06%
|
5.0
|
280.6
|
1.217
|
200
|
10%
|
1.94
|
109
|
1.09
|
515.5
|
Ammonium hydroxide
|
NH4
OH
|
35.0
|
28%
|
14.8
|
251
|
0.898
|
67.6
|
Acetic acid, glacial
|
CH3
COOH
|
60.05
|
99.5%
|
17.4
|
1045
|
1.05
|
57.5
|
Acetic acid
|
36%
|
6.27
|
376
|
1.045
|
159.5
|
Formic acid
|
HCOOH
|
46.02
|
90%
|
23.4
|
1080
|
1.20
|
42.7
|
Hydrochloric acid
|
HCl
|
36.5
|
36%
|
11.6
|
424
|
1.18
|
86.2
|
10%
|
2.9
|
105
|
1.05
|
344.8
|
Nitric acid
|
HNO3
|
63.02
|
71%
|
15.99
|
1008
|
1.42
|
62.5
|
67%
|
14.9
|
938
|
1.40
|
67.1
|
61%
|
13.3
|
837
|
1.37
|
75.2
|
Perchloric acid
|
HClO4
|
100.5
|
70%
|
11.65
|
1172
|
1.67
|
85.8
|
60%
|
9.2
|
923
|
1.54
|
108.7
|
Phosphoric acid
|
H3
PO4
|
98.0
|
85%
|
18.1
|
1445
|
1.71
|
55.2
|
Sulfuric acid
|
H2
SO4
|
98.1
|
96%
|
18.0
|
1766
|
1.84
|
55.6
|
|
NaOH
Mw=40g/M; (store at NT).
|
Conc.
|
Stock
|
%(w/w)
|
50ml
|
200ml
|
300ml
|
|
NaOH
|
10M
|
40g/M
|
30.1
|
20g
|
80g
|
120g
|
|
H2
O
|
|
mQ
|
69.90
|
46.45ml
|
185.8ml
|
278.7ml
|
|
|
10M: 30.10%; p=1.329.
|
Conc.
|
Stock
|
%(w/w)
|
50ml
|
150ml
|
200ml
|
|
NaOH
|
1M
|
10M
|
12.87
|
5ml
6.645g
|
15ml
19.94g
|
20ml
26.58g
|
|
H2
O
|
|
mQ
|
87.13
|
45ml
|
135ml
|
180ml
|
|
|
- it is better to use plastic bottles for storage, because the alkali slightly solubilize the glass.
KOH
Mw=56.11g/M; (store at NT).
|
Conc.
|
Stock
|
%(w/w)
|
10ml
|
50ml
|
100ml
|
150ml
|
|
KOH
|
5M
|
56.11g/M
|
23.05
|
2.806g
|
14.03g
|
28.06g
|
42.08g
|
|
H2
O
|
|
mQ
|
76.95
|
9.363g
|
46.81g
|
93.63g
|
140.4g
|
|
|
5M: 23.06%; p=1.217.
- it is better to use plastic bottles for storage, because the alkali slightly solubilize the glass.
TCA
CCl3
CO2
H Mw=163.39g/M,(store at NT, in the dark, under the fume hood).
|
Conc.
|
%(w/w)
|
5ml
|
10ml
|
25ml
|
50ml
|
|
TCA
|
100% (w/v)
|
68.78
|
5.0g
|
10.0g
|
25.0g
|
50.0g
|
|
H2
O
|
mQ
|
31.22
|
2.27ml
|
4.54ml
|
11.35ml
|
22.7ml
|
|
|
Detergents.
Detergent
|
Tmelt
|
Mw [Da]
|
CMC
|
monomer
|
micelle
|
%(w/v)
|
M
|
Anionic
|
SDS
|
206
|
288
|
18,000
|
0.23
|
8.0 x 10-3
|
Cholate
|
201
|
430
|
4,300
|
0.60
|
1.4 x 10-2
|
Deoxycholate
|
175
|
432
|
4,200
|
0.21
|
5.0 x 10-3
|
Cationic
|
C16
TAB
|
230
|
365
|
62,00
|
0.04
|
1.0 x 10-3
|
Amphoteric
|
LysoPC
|
-
|
495
|
92,000
|
0.0004
|
7.0 x 10-6
|
CHAPS
|
157
|
615
|
6,150
|
0.49
|
1.4 x 10-3
|
Zwittergent 3-14
|
-
|
365
|
30,000
|
0.011
|
3.0 x 10-4
|
Nonionic
|
Octyl glucoside
|
105
|
292
|
8,000
|
0.73
|
2.3 x 10-2
|
Digitonin
|
235
|
1,229
|
70,000
|
-
|
-
|
C12
E8
|
-
|
542
|
65,000
|
0.005
|
8.7 x 10-5
|
Lubrol PX
|
-
|
582
|
64,000
|
0.006
|
1.0 x 10-4
|
Triton X-100
|
-
|
650
|
90,000
|
0.021
|
3.0 x 10-4
|
Nonident P-40
|
-
|
603
|
90,000
|
0.017
|
3.0 x 10-4
|
Tween 80
|
-
|
1,310
|
76,000
|
0.002
|
1.2 x 10-5
|
|
CMC - critical micelle concentration.
Detergent
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
11
|
12
|
1
- Strongly denaturing;
2
- Dializable;
3
- Ion exchangeable, unsuitable for ion-exchange chromatography;
4
- Complexes ions;
5
- Strong A280
;
6
- Assay interference;
7
- Cold precipitates;
8
- High cost;
9
- Ease of purification;
10
- Radiolabeled;
11
- Definite composition;
12
- Auto-oxidation
|
Anionic
|
SDS
|
+
|
+
|
+
|
+
|
-
|
-
|
+
|
-
|
+
|
+
|
+
|
-
|
Cholate
|
-
|
+
|
+
|
+
|
-
|
-
|
-
|
-
|
+
|
+
|
+
|
-
|
Deoxycholate
|
-
|
+
|
+
|
+
|
-
|
-
|
+
|
-
|
+
|
+
|
+
|
-
|
Cationic
|
C16
TAB
|
+
|
+
|
+
|
-
|
-
|
-
|
+
|
-
|
+
|
-
|
+
|
-
|
Amphoteric
|
LysoPC
|
+/-
|
-
|
-
|
-
|
-
|
-
|
-
|
+
|
+/-
|
+
|
+
|
-
|
CHAPS
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
+
|
+
|
-
|
+
|
-
|
Zwittergent 3-14
|
+/-
|
+/-
|
-
|
-
|
-
|
-
|
-
|
+
|
+
|
-
|
+
|
-
|
Nonionic
|
Octyl glucoside
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
+
|
-
|
+
|
+
|
-
|
Digitonin
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
+
|
+
|
-
|
|
|
C12
E8
|
-
|
-
|
-
|
+/-
|
-
|
-
|
-
|
+
|
-
|
+
|
-
|
+
|
Lubrol PX
|
-
|
-
|
-
|
+/-
|
-
|
+/-
|
-
|
-
|
-
|
+
|
-
|
+
|
Triton X-100
|
-
|
-
|
-
|
+/-
|
+
|
+/-
|
-
|
-
|
-
|
+
|
-
|
+
|
Nonident P-40
|
-
|
-
|
-
|
+/-
|
+
|
+/-
|
-
|
-
|
-
|
+
|
-
|
+
|
Tween 80
|
-
|
-
|
-
|
+/-
|
-
|
+/-
|
-
|
-
|
-
|
+
|
-
|
+
|
|
C16
TAB - hexadecyl trimethylammonium bromide;
LysoPC - lysophosphatidylcholine. ~undefined Sodium cholate and sodium deoxycholate are unsoluble at <pH 7.5 or at ionic strength greater then 0.1%. SDS may precipitate below 20o
C; ~undefined_Ionic_detergents_may_induce_problems_with_electrophoresis_and_isoelectric_focusing~J~Kbr_~H~M~undefined*_It_is_possible_to_remove_by_dialysis~J~Kbr_~H~M~undefined**_Phenol_~Fcontaining_detergents_~Afor_example_Triton_X~F100_and_NP~F40~B_precipitate_during_Folin_protein_assay_~Abut_do_not_interfere_with_Bradford_protein_assay~B._~Kbr_~H~M~1~Kbr_~H~M~1~K~Hfont~M~1~1~Ka_name~L~4a29~4~M~K~Ha~M~1~1~Kp_class~L~4h3~4~M~Kfont~MN~FLauroylsarcosine_Na~K~Hfont~M~1~K~Hp~M~1~1~Kp~M~Kfont~M~Astore_at_NT~B.~K~Hfont~M~1~K~Hp~M~1~1~1~1~1~Ktable~M~Ktbody~M~Ktr~M~Ktd~M~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~1~Ktable~M~Ktbody~M~Ktr~M~Ktd~M~1~Kfont~M~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~MConc.~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~MStock~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M~7~Aw~Hw~B~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M1ml~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M3ml~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M5ml~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M15ml~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M50ml~K~Hfont~M~1~K~Htd~M~1~Ktd~M~1~Kfont~M~K~Hfont~M~1~K~Htd~M~1~K~Htr~M~1~Ktr~M~Ktd~M~Kfont~MSarc~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M10~7~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M30~7~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M33.93~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M~Kfont~M~Ku~M0.333ml~K~Hu~M~1~Kbr_~H~M~10.342g~K~Hfont~M~1~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M~Kfont~M~Ku~M1.00ml~K~Hu~M~1~Kbr_~H~M~11.027g~K~Hfont~M~1~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M~Kfont~M~Ku~M1.67ml~K~Hu~M~1~Kbr_~H~M~11.712g~K~Hfont~M~1~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M~Kfont~M~Ku~M5.00ml~K~Hu~M~1~Kbr_~H~M~15.14g~K~Hfont~M~1~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M~Kfont~M~Ku~M16.67ml~K~Hu~M~1~Kbr_~H~M~117.12g~K~Hfont~M~1~K~Hfont~M~1~K~Htd~M~1~Ktd~M~1~Kbr_~H~M~1~1~Kfont~M~K~Hfont~M~1~K~Htd~M~1~K~Htr~M~1~Ktr~M~Ktd~M~Kfont~MH~Ksub~M2~K~Hsub~M~1O~K~Hfont~M~1~K~Htd~M~1~Ktd~M~Kfont~M
|
mQ
|
66.07
|
0.667ml
|
2.00ml
|
3.33ml
|
10.00ml
|
33.33ml
|
|
p=1.017.
SDS
Sodium dodecyl sulfate, sodium lauryl sulfate; [CH3
(CH2
)10
CH2
SO4
]Na; Mw=288.4g/M; (store at NT).
|
Conc.
|
Stock
|
%(w/w)
|
100 ml
|
250 ml
|
300 ml
|
400 ml
|
|
SDS
|
10%(w/v)
|
solid
|
9.82
|
10g
|
25g
|
30g
|
40g
|
|
H2
O
|
|
mQ
|
90.18
|
91.8 ml
|
229.5 ml
|
275.4 ml
|
367.2 ml
|
|
|
p = 1.018
weight under the fume hood (and better to wear the mask);
it is necessary to heat to 60-80o
C. to facilitate solubilization. Check pH. If it differs from neutral (~7.2-7.5) dramatically - adjust by diluted alkali / acid.
Organic solvents.
Phenol
C6
H5
OH; Mw=94.1g/M
p=1.054, tm
= 43, tb
= 182, pKa=10.0
solubility: 6.816
H2
O, unlimited 66
H2
O; unlimited EtOH
Preparation of "acidic" and "neutral" phenol.
a) from the distillation:
- distill phenol under H2
O;
- adjust water to the volume of about 1/10 of phenol phase;
- add 8-hydroxyquinoline to 0.1% (relative to phenol phase) and bMeEtOH (2-mercaptoethanol) to 0.2% (relative to H2
O = 0.02% relative to phenol phase);
---- on this step you obtained the "acidic phenol". Store at -20o
C (~1 year); ----
- add about the same volume of 0.2 M Tris-base to the phenol, mix ~0.5-1h;
- throw away aqueous phase;
- + 0.1V 0.1M Tris-Cl, pH 8.0
- 0.2% bMeEtOH
- mix ~0.5-1h;
- store at 4o
C (in the dark) ~several months.
b) from the good commercial substance:
- saturate phenol by the water (add about 1/5 V );
- then, according to (a), from the p.2.
______________
~undefined For RNA extraction it is better to saturate (just add) "acidic phenol" by the following buffer: (store at 4o
C):
|
Conc.
|
Stock
|
100ml
|
|
AcONa, pH 5.1
|
50mM
|
3M
|
1.67ml
|
|
EDTA
|
10mM
|
0.5M
|
2.0ml
|
|
H2
O
|
|
mQ
|
96.0ml
|
|
|