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        单组分溶液配制

        互联网

        1941

         

        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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