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    单组分溶液配制

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    1768

     

    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>
     

    Guanidine HCl (GuHCl)

    CH5 N3 xHCl, Mw=95.53g/M

     

    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

    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.

    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~2~1~0~0~0~undefined*_It_is_possible_to_remove_by_dialysis~J~Kbr_~H~M~2~1~0~0~0~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~2~1~0~0~0~0~Kbr_~H~M~2~1~0~0~0~0~K~Hfont~M_~2~1~0~0~0~0~K~Ha~M~Ka_name~L~4a28~4~M~2~1~0~0~0~0~0~K~Ha~M~Ka_name~L~4a28~4~M~K~Ha~M~Ka_name~L~4a29~4~M_~2~1~0~0~0~0~0~K~Ha~M~Kp_class~L~4h3~4~M~Ka_name~L~4a29~4~M~2~1~0~0~0~0~0~0~K~Ha~M~Ka_name~L~4a29~4~M~Kfont~MN~FLauroylsarcosine_Na~K~Hfont~M_~K~Ha~M~K~Hp~M~Ka_name~L~4a29~4~M~2~1~0~0~0~0~0~K~Ha~M~Kp~M~Ka_name~L~4a29~4~M~2~1~0~0~0~0~0~0~K~Ha~M~Ka_name~L~4a29~4~M~Kfont~M~Astore_at_NT~B.~K~Hfont~M_~K~Ha~M~K~Hp~M~Ka_name~L~4a29~4~M~2~1~0~0~0~0~0~Ktable~M~2~1~0~0~0~0~0~0~Ktbody~M~2~1~0~0~0~0~0~0~0~Ktr~M~2~1~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~Ktable~M~2~1~0~0~0~0~0~0~0~0~0~0~Ktbody~M~2~1~0~0~0~0~0~0~0~0~0~0~0~Ktr~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M_~Kfont~M_~K~Hfont~M~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~MConc.~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~MStock~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M~7~Aw~Hw~B~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M1ml~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M3ml~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M5ml~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M15ml~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M50ml~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M_~Kfont~M_~K~Hfont~M~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~K~Htr~M~2~1~0~0~0~0~0~0~0~0~0~0~0~Ktr~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~MSarc~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M10~7~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M30~7~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M33.93~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M~Kfont~M~Ku~M0.333ml~K~Hu~M~Kbr_~H~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~00.342g~K~Hfont~M_~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M~Kfont~M~Ku~M1.00ml~K~Hu~M~Kbr_~H~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~01.027g~K~Hfont~M_~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M~Kfont~M~Ku~M1.67ml~K~Hu~M~Kbr_~H~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~01.712g~K~Hfont~M_~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M~Kfont~M~Ku~M5.00ml~K~Hu~M~Kbr_~H~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~05.14g~K~Hfont~M_~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~M~Kfont~M~Ku~M16.67ml~K~Hu~M~Kbr_~H~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~017.12g~K~Hfont~M_~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kbr_~H~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0_~Kfont~M_~K~Hfont~M~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~K~Htr~M~2~1~0~0~0~0~0~0~0~0~0~0~0~Ktr~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~M~Kfont~MH~Ksub~M2~K~Hsub~M_O~K~Hfont~M_~K~Ha~M~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~Ka_name~L~4a29~4~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

    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

     

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