Single component solutions.

About the recalculation of recipes for the arbitrary volume:

Organic substances.
pK_a and temperature dependence of pH for common buffers.
Buffer Mw pKa 20oC Working range delta pKa per 10oC 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   Bis-Tris Propane 1,3-bis[tris(hydroxymethyl)methylamino]propane 282.3 6.8* 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

* pKa=9.0 for the second stage of dissociation.
ATP
C₁₀H₁₄N₅O₁₃P₃Na₂ Mw=551.1 g/M; (store at -20^oC).
Conc. Stock 1ml ATP 0.1M 551.1g/M 55.1mg H2O   mQ
0.1M ATP (pH 7.2): 5.71% (w/w) ATP, 84.86% (w/w) H₂O, 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=A_{259 1:2000}x0.130; then, adjust the final volume.
Betaine
monohydrate C₅H₁₁NO₂xH₂O; Mw=135.2g/M; (store at 4^oC).
Conc. Stock %(w/w) 100ml 200ml 700ml Betaine 135.2g/M 5M 63.00 67.6g 135.2g 473.2g H2O   mQ 37.00 39.74ml 79.49ml 278.2ml
p~1.073g/ml
Cresol red
50mM (store at +4, -20^oC):
Conc. Stock 1ml 50ml cresol red 50mM 404.4g/M 20.2mg 1.01g H2O   mQ
Cresol Red 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 <7.5). 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
C₄H₁₀O₂S₂ Mw=154.2g/M; (store at -20^oC).
Conc. Stock %(w/w) 1ml 5ml 10ml 20ml DTT 154.2g/M 1M 14.55 0.155g 0.773g 1.55g 3.09g H2O   mQ 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 H2O   mQ
filter sterilization;
no DEPC treatment;
we do not know why, but many manuals (probably, after "Maniatis") recommend to prepare it in 10mM AcNa, pH 5.2.
Preparation of dNTP's.
The quick protocol for ~100mM stock.
1. dilute all four dNTP's (250mg of each) in 3.676ml of H₂O;
2. + 424µl 5M NaOH;
3. check the pH: ~0.5µl on pH-paper.


Normal (accurate) protocol for 100mM stocks preparation.
1. add the necessary quantity (see table) of H₂O 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 C10H14N5O12P3Na2x3H2O 589.2 3.24ml 850 µl 4.24ml dGTP C10H13N5O13P3Na3x2H2O 609.2 3.55ml 400 µl 4.10ml dCTP C9H13N3O13P3Na3x2H2O 569.1 3.44ml 800 µl 4.39ml dTTP C10H14N2O14P3Na3x2H2O 584.1 3.73ml 400 µl 4.28ml

2. check the pH: ~0.5µl on pH-paper;
3. 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 A_m~0.3 - the most accurate range for spectrophotometer).
   Concentration is c[mM]=k_1:5000xA_m.
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]=A_max/E; A_max = maximum of absorption.
EDTA
C₁₀H₁₄O₈N₂Na₂x2H₂O; Mw=372.3g/M; pH = 8.0 (store at 4^oC).
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 H2O   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 H2O   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.
not to treat by DEPC.
EtBr
Ethidium bromide, C₂₁H₂₀N₃Br; Mw=394.3g/M; (store at NT in the dark);
Conc. 5ml 10ml 50ml EtBr 10mg/ml 50mg 100mg 500mg
soluble in H₂O, 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 4^oC).
Conc. Stock 10ml 50ml 100ml Gelatin 2% solid 0.2g 1.0g 2.0g H2O   mQ
autoclave sterilization.
Glucose
C₆H₁₂O₆xH₂O, Mw=198.17g/M;

Conc. Stock %(w/w) 50ml 100ml 250ml C6H12O6xH2O 2M 198.17g/M 34.972 19.82g 39.63g 99.09g H2O   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 H2O [ml] 868.23 802.745 736.96 %(w/w) 18.583 27.023 34.972 H2O %(w/w) 81.417 72.977 65.028 p (g/ml) 20oC 1.0664 1.1 1.1333
Guanidine HCl
CH₅N₃xHCl, 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 H2O (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 H2O %(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 25^oC -> 8.54M, 5^oC->>8M;
A₂₆₀(6M in H₂O)<0.03;
it is possible to take the "partial density of Guanidine HCl" as 0.763 in calculations of solutions.
HEPES
Conc. Stock %(w/w) 1L C8H18N2O4S 1M 238.3g/M 22.40 238.3g H2O   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
C₃H₄N₂, (store at 4^oC):
Conc. Stock 50ml 100ml Imidazole 2M 68.1g/M 6.81g 13.62g H2O   mQ
Paraformaldehyde
PFA (paraformaldehyde) 37% (for histochemistry it should be freshly prepared).
1. mix in the screw-cap tube:
PFA (solid) => 0.37g,
H₂O => 1.0ml
NaOH (1N) => 14µl;
2. solubilize in the boiling water bath (to heat until pH will drop to ~7.0 =>~1-3').
PEG
H(OCH₂CH₂)_nOH; (store at 4^oC).
Conc. %(w/w) 10ml 50ml 100ml 150ml 200ml PEG6000 40% 37.21 4.0g 20g 40g 60g 80g H2O mQ 62.79 6.75g 33.75g 67.5g 101.25g 135.0g
p=1.075.
PMSF
(store at -20^oC)
Conc. Stock 20ml C7H7FO2S 100mM 174.2g/M 0.348g Isopropanol     20ml
Retinoic acid
(all trans-R.a., Tretinoin, light-sensitive, store at -20^oC):
Conc. Stock 16.6ml C20H28O2 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
C₁₂H₂₂O₁₁, Mw=342.30g/M; 20^oC.

Conc. Stock %(w/w) 50ml 100ml 250ml Sucrose 1M 342.30g/M 30.330 17.115g 34.23g 85.58g H2O   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 H2O [ml] 786.3 570.4 460.35 %(w/w) 30.330 54.550 65.022 H2O %(w/w) 69.670 45.450 34.978 p (g/ml) 18oC 1.1286 1.2550 1.3161
Tris Cl
C₄H₁₁O₃N; Mw=121.1g/M; (store at 4^oC).
Conc. Stock 50 ml 100 ml 150ml 200ml Tris-base 1M 121.1g/M 6.06 12.11g 18.17g 24.22g H2O 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) H₂O; p=1.058
not to treat by DEPC;
autoclave sterilization;
pH of Tris-buffers is dependent from concentration. If to take as original the 50mM solution:
pH(500mM) => + 0.05
pH(5mM) => - 0.05
pH drops on 0.028 when the temperature rise on 1^oC.
Temperature dependence of pH for Tris Cl 50mM.
pH at g/50ml 1M or g/liter for 0.05 M 5°C 25°C 37°C Tris HCl Tris Base H2O Tris HCl Tris Base H2O 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
C₆H₁₃NO₅, Mw=179.2g/M;(store at 4^oC).
Conc. Stock 50ml Tricine 1M 179.2g/M 8.96g H2O   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 4^oC):
Conc. Stock %(w/w) 50ml Triethanolamine 1M 149.19g/M 14.69 7.46g 6.66ml H2O   mQ 85.31 43.34ml
Urea
CH₄N₂O, Mw=60.06g/M;
Molarity; 1000ml / 1M 2M 3M 4M 5M 6M 7M 8M 9M 10M CH4N2O [g] 60.06 120.12 180.18 240.24 300.30 360.36 420.42 480.48 540.54 600.60 H2O [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 CH4N2O %(w/w) 5.94 11.71 17.30 22.72 28.02 33.15 38.16 43.05 47.81 52.47 H2O %(w/w) 94.06 88.29 82.70 77.28 71.98 66.85 61.84 59.95 52.19 47.53
solubility: at 25^oC: 10.49M, 5^oC: ~8M;
A₂₆₀(6M in H₂O)<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 NH4OH 35.0 28% 14.8 251 0.898 67.6 Acetic acid, glacial CH3COOH 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 H3PO4 98.0 85% 18.1 1445 1.71 55.2 Sulfuric acid H2SO4 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 69.90 20g 80g 120g H2O   mQ 30.1 46.45ml 185.8ml 278.7ml

10M: 30.10%; p=1.329.

Conc. Stock 50ml 150ml 200ml NaOH 1M 10M 5ml 6.645g 15ml 19.94g 20ml 26.58g H2O   mQ 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 H2O   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.

Trichloroacelic acid

Trichloroacelic acid,CCl₃CO₂H Mw=163.39, (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 H2O 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 C16TAB 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 - - C12E8 - 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 - ÈIon 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 C16TAB + + + - - - + - + - + - Amphoteric LysoPC +/- - - - - - - + +/- + + - CHAPS - + - - - - - + + - + - Zwittergent 3-14 +/- +/- - - - - - + + - + - Nonionic Octyl glucoside - + - - - - - + - + + - Digitonin - - - - - - - + + -     C12E8 - - - +/- - - - + - + - + Lubrol PX - - - +/- - +/- - - - + - + Triton X-100 - - - +/- + +/- - - - + - + Nonident P-40 - - - +/- + +/- - - - + - + Tween 80 - - - +/- - +/- - - - + - +

C₁₆TAB - hexadecyl trimethylammonium bromide;
LysoPC - lysophosphatidylcholine.
* Sodium cholate and sodium deoxycholate are unsoluble at <pH 7.5 or at ionic strength greater then 0.1%. SDS may precipitate below 20^oC.
** Ionic detergents may induce problems with electrophoresis and isoelectric focusing.
*** It is possible to remove by dialysis.
**** Phenol -containing detergents (for example Triton X-100 and NP-40) precipitate during Folin protein assay (but do not interfere with Bradford protein assay).



N-Lauroylsarcosine Na

(store at NT).

Conc. Stock 1ml 3ml 5ml 15ml 50ml Sarc 10% 30% 0.333ml 0.342g 1.00ml 1.027g 1.67ml 1.712g 5.00ml 5.14g 16.67ml 17.12g H2O   mQ 0.667ml 2.00ml 3.33ml 10.00ml 33.33ml

p=1.017.

SDS

Sodium dodecyl sulfate, sodium lauryl sulfate; [CH₃(CH₂)₁₀CH₂SO₄]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 H2O   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 60-80^oC. to facilitate solubilization. Check pH. If it differs from neutral (~7.2-7.5) dramatically - adjust by diluted alkali / acid.

Organic solvents.

Phenol

C₆H₅OH; Mw=94.1g/M
p=1.054, t_m= 43, t_b= 182, pKa=10.0
solubility: 6.8¹⁶H₂O, unlimited ⁶⁶H₂O; unlimited EtOH

Preparation of "acidic" and "neutral" phenol.

a) from the distillation:

1. distill phenol under H₂O;
2. adjust water to the volume of about 1/10 of phenol phase;
3. add 8-hydroxyquinoline to 0.1% (relative to phenol phase) and bMeEtOH (2-mercaptoethanol) to 0.2% (relative to H₂O = 0.02% relative to phenol phase);

---- on this step you obtained the "acidic phenol". Store at -20^oC (~1 year); ----

4. add about the same volume of 0.2 M Tris-base to the phenol, mix ~0.5-1h;
5. throw away aqueous phase;
6. + 0.1V 0.1M Tris-Cl, pH 8.0
7. 0.2% bMeEtOH
8. mix ~0.5-1h;
9. store at 4^oC (in the dark) ~several months.

b) from the good commercial substance:

1. saturate phenol by the water (add about 1/5 V );
2. then, according to (a), from the p.2.

______________
* For RNA extraction it is better to saturate (just add) "acidic phenol" by the following buffer: (store at 4^oC):

Conc. Stock 100ml AcONa, pH 5.1 50mM 3M 1.67ml EDTA 10mM 0.5M 2.0ml H2O   mQ 96.0ml

check pH and adjust by diluted (1:100) acetic acid if necessary.

there was note in the "Mitchell Group Methods" that acidic phenol is much more stable, then the neutral. They recommend preparing neutral phenol in small quantities only for a few weeks.

EtOH

CH₃CH₂OH; Mw=46.1g/M;

p (w/w) % (v/v) %   p (w/w) % (v/v) %   p (w/w) % (v/v) % 0.7892 100.00     0.83 85.4     0.89 60.5   0.79 99.7     0.84 81.4     0.90 56.12   0.80 96.5     0.85 77.3     0.91 51.71   0.8042 95.00 96.80   0.86 73.2     0.92 47.2   0.81 92.9     0.8678 70.02 76.96   0.93 42.50   0.8114 92.42 95.01   0.87 69.0     0.94 37.6   0.8178 90.05 93.31   0.88 64.8           0.82 89.2     0.8855 62.40 70.01

very hygroscopic, (store at NT in tightly closed bottle); t_melt.=-117.3^oC; t_boil.=78.5^oC.




Conc. Stock 300ml EtOH 70% 96% 190g H2O   mQ 70ml

Preparation of 100% EtOH.

1. dry CuSO₄ (at 65^oC, 1-2 days) to the practically white color;
2. mix about 1/4 of volume of the CuSO₄ with 3/4 volume of 96%EtOH;
3. mix intensively;
4. leave for 1-2 days (CuSO₄ will "take" water from ethanol).

test for the "100%" ethanol: add a drop of ethanol to the xylene. No mist should appear in the case of 100% ethanol.

"Practical Molecular Biology" http://molbiol.edu.ru
e-mail: pmb@molbiol.edu.ru