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单组分溶液配制

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摘要：BufferMwpKa20oCWorkingrangedeltapKaper10oCMES2-(N-morpholino)ethanesulfonicacid195。110PIPESpiperasine-N,N‘-bis(2-ethanesulfonicacid)302。085ACES2-[(2-amino-2-oxoethyl)amino]ethanesulfonicacid182。5BESN,N-bis(2-hydroxyethyl)-2-aminoethanesulfonicacid213。...

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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".




    About the recalculation of recipes for the arbitrary volumes:


    Organic substances.

    pKa 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  
    Imidazol - HCl 68.08 6.95 6.2-7.8  
    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 -20oC).

    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.

    1. adjust to pH7.5 by 2M NaOH (~70-80µl);
    2. 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 1:2000x0.130

    3. adjust the final volume.

    Betaine

    monohydrate C5H11NO2xH2O; Mw=135.2g/M; (store at 4oC).

    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 (Na)

    50mM (store at +4, -20oC):

    Conc. Stock 1ml 50ml
    Cresol red (Na) 50mM 404.4g/M 20.2mg 1.01g
    H2O   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

    C4H10O2S2 Mw=154.2g/M; (store at -20oC).

    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.

    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.

    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 Am~0.3 - the most accurate range for spectrophotometer).
      Concentration is c[mM]=k1:5000xAm.

    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

    C10H14O8N2Na2x2H2O; Mw=372.3g/M; pH = 8.0 (store at 4oC).

    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;
  • do not 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 4oC).

    Conc. Stock 10ml 50ml 100ml
    Gelatin 2% solid 0.2g 1.0g 2.0g
    H2O   mQ        
  • sterilize by autoclaving.

    • Glucose

    C6H12O6xH2O, Mw=198.17g/M (store at 4oC)
    Conc. Stock %(w/w) 50ml 100ml 250ml
    Glucose 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 (GuHCl)

    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 25oC - 8.54M, 5oC - >8M;
  • A260(6M in H2O)<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

    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 4oC):

    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 ~1-3'; until pH will drop to ~7.0).

    PEG

    H(OCH2CH2)nOH; (store at 4oC).

    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 -20oC)

    Conc. Stock 20ml
    C7H7FO2S 100mM 174.2g/M 0.348g
    Isopropanol     20ml

    Retinoic acid

    all trans-Retinoic acid, Tretinoin, light-sensitive, (store at -20oC):

    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; 20oC.

    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
    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 4oC).

    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

  • 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 1oC.

    • Temperature dependence of pH for Tris Cl 50mM.

    pH at g/50ml 1M or
    g/liter for 0.05 M    		    
    5oC 25oC 37oC 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 4oC).

    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 4oC):

    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 /
    1 2 3 4 5 6 7 8 9 10

    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 25oC: 10.49M, 5oC: ~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 30.1 20g 80g 120g
    H2O   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
    H2O   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
    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.

    • TCA

    CCl3CO2H 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
    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 - 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
    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 20oC;
    ** 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 %(w/w) 1ml 3ml 5ml 15ml 50ml
    Sarc 10% 30% 33.93 0.333ml
    0.342g    		 1.00ml
    1.027g    		 1.67ml
    1.712g    		 5.00ml
    5.14g    		 16.67ml
    17.12g
    H2O   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)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 60-80oC. 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, tm= 43, tb= 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 -20oC (~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 4oC (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 4oC):

    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;

    densities of aquatic solutions of ethanol.
  • very hygroscopic, (store at NT in tightly closed bottle); tпл.=-117.3oC; tкип.=78.5oC.
    Conc. Stock 300ml
    EtOH 70% 96% 190g
    H2O   mQ 70ml

    Preparation of 100% EtOH.

    1. dry CuSO4 (at 65oC, 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.

作者： 2008-2-3
医学百科App—中西医基础知识学习工具
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