丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

单组分溶液配制

互联网

5805

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.

    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 1:2000 x0.130

    3. 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.

    1. dilute all four dNTP's (250mg of each) in 3.676ml of H2 O;
    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 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
    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]=k1:5000 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:

    1. distill phenol under H2 O;
    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 H2 O = 0.02% relative to phenol phase);

      ---- on this step you obtained the "acidic phenol". Store at -20o C (~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 4o C (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.

    ______________
    ~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

    提问
    扫一扫
    丁香实验小程序二维码
    实验小助手
    丁香实验公众号二维码
    扫码领资料
    反馈
    TOP
    打开小程序