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Single component solutions.




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Ñòðóêòóðíàÿ îðãàíèçàöèÿ áåëêà
Å.Ì.Ïîïîâ
Rambler's Top100
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.

    BufferMwpKa
    20oC    		Working
    range    		delta pKa
    per 10oC
    MES
    2-(N-morpholino)ethanesulfonic acid    		195.26.155.5-6.7-0.110
    Bis-Tris
    bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane    		209.26.55.8-7.2 
    ADA
    N-(2-acetamido)-2-imidoacetic acid    		190.26.606.0-7.2-0.110
    PIPES
    piperasine-N,N'-bis(2-ethanesulfonic acid)    		302.46.806.1-7.5-0.085
    ACES
    2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid    		182.26.906.1-7.5-0.200
    MOPSO
    3-(N-morpholino)-2-hydroxypropanesulfonic acid    		225.36.96.2-7.6 
    Imidazol - HCl68.086.956.2-7.8 
    Bis-Tris Propane
    1,3-bis[tris(hydroxymethyl)methylamino]propane    		282.36.8*6.3-9.5 
    BES
    N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid    		213.27.156.4-7.8-0.160
    MOPS
    3-(N-morpholino)propanesulfonic acid    		209.37.206.5-7.9-0.013
    TES
    N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid    		229.27.506.8-8.2-0.200
    HEPES
    N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)    		238.37.556.8-8.2-0.140
    DIPSO
    3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxyprpanesulfonic acid    		243.37.67.0-8.2 
    TAPSO
    3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid    		259.37.67.0-8.2 
    HEPPSO
    N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)    		268.37.87.1-8.5 
    POPSO
    piperazine-N,N'-bis(2-hydroxypropanesulfonic acid)    		362.47.87.2-8.5 
    TEA
    triethanolamine    		149.27.87.3-8.3 
    EPPS
    N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)    		252.38.07.3-8.7 
    Tricine
    N-tris(hydroxymethyl)methylglycine    		179.28.157.4-8.8-0.210
    Tris (TRIZMA)
    tris(hydroxymethyl)aminomethane    		121.18.307.0-9.1-0.310
    Bicine
    N,N-bis(2-hydroxyethyl)glycine    		163.28.357.6-9.0-0.180
    TAPS
    N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid    		243.38.47.7-9.1 
    Glycylglycine 8.40 -0.280
    AMPSO
    3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid    		227.39.08.3-9.7 
    CHES
    1-(N-cyclohexylamino)ethanesulfonic acid    		207.39.38.6-10.0 
    CAPSO
    3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid    		237.39.68.9-10.3 
    AMP
    2-amino-2-methyl-1-propanol    		89.19.79.0-10.5 
    CAPS
    3-(cyclohexylamino)-1-propanesulfonic acid    		221.310.49.7-11.1 

    * pKa=9.0 for the second stage of dissociation.

    ATP

    C10H14N5O13P3Na2 Mw=551.1 g/M; (store at -20oC).

    Conc.Stock1ml
    ATP0.1M551.1g/M55.1mg
    H2O mQ  

    Unchecked information 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:

      3. 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)100ml200ml700ml
    Betaine135.2g/M5M63.0067.6g135.2g473.2g
    H2O mQ37.0039.74ml79.49ml278.2ml

    p~1.073g/ml

    Cresol red (Na)

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

    Conc.Stock1ml50ml
    Cresol red (Na)50mM404.4g/M20.2mg1.01g
    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)1ml5ml10ml20ml
    DTT154.2g/M1M14.550.155g0.773g1.55g3.09g
    AcNa, pH 5.2 10mM85.45905 µl4.53ml9.05ml18ml

    p~1.06g/ml

    Conc.Stock1ml5ml10ml20ml
    DTT154.2g/M2.2M0.339g1.696g3.394g6.787g
    AcNa, pH 5.2 10mM     
  • filter sterilization;
  • 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);
      2.    

      Mw

      		V(H2O)V(Tris base 1M)V(final)
      dATPC10H14N5O12P3Na2x3H2O589.23.24ml850 µl4.24ml
      dGTPC10H13N5O13P3Na3x2H2O609.23.55ml400 µl4.10ml
      dCTPC9H13N3O13P3Na3x2H2O569.13.44ml800 µl4.39ml
      dTTPC10H14N2O14P3Na3x2H2O584.13.73ml400 µl4.28ml
    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:

      MwAmpHEk (for 1:5000)
    dATP[Na2]589.22597.015.2x103328.9
    dGTP[Na3]609.22537.513.7x103365.0
    dCTP[Na3]569.12802.0 (!!!)13.0x103384.6
    dTTP[Na3]584.12677.59.6x103520.8
           
    ATP [Na4]595.1259 15.4x103 
    CTP [Na4]571.1280 13.0x103 
    GTP [Na4]611.1252 13.7x103 
    UTP [Na4]572.1262 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 ml100 ml150 ml250 ml
    EDTA0.5 M372.3g/M16.989.31g18.62g27.92g46.55g
    NaOH~0.5 M40g/M1.821.014g2.028g3.042g5.07g
    H2O mQ81.1944.48ml88.95ml133.4ml222.4ml
    Conc.Stock10 ml50 ml100 ml150 ml250 ml
    EDTA0.5 M372.3g/M1.86g9.31g18.62g27.92g46.55g
    NaOH~0.5 M10M 507µl
    0.674g    		2.535ml
    3.37g    		5.07ml
    6.74g    		7.61ml
    10.11g    		12.68ml
    16.85g
    H2O mQ8.42ml42.12ml84.24ml126.4ml210.6ml

    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.5ml10ml50ml
    EtBr10mg/ml50mg100mg500mg
  • 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.Stock10ml50ml100ml
    Gelatin2%solid0.2g1.0g2.0g
    H2O mQ    
  • autoclave sterilization.

    • Glucose

    C6H12O6xH2O, Mw=198.17g/M (store at 4oC)
    Conc.Stock%(w/w)50ml100ml250ml
    Glucose2M198.17g/M34.97219.82g39.63g99.09g
    H2O mQ65.02836.85ml73.70ml184.24ml
    1000ml/
    198.17g/M1M1.5M2M
    Glucose [g]198.17
    		297.255
    		396.34
    H2O [ml]868.23
    		802.745
    		736.96
    %(w/w)18.58327.02334.972
    H2O %(w/w)81.41772.97765.028
    p (g/ml) 20oC1.06641.11.1333

    Guanidine HCl (GuHCl)

    CH5N3xHCl, Mw=95.53g/M

    Molarity; 1000ml /
    12345678
    GuHCl95.53g
    		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)9.3718.2826.7834.9342.7550.2657.5064.50
    H2O %(w/w)90.6381.7273.2265.0757.2549.7442.5035.50
    p (g/ml)1.0201.0451.0701.0941.1171.1401.1631.185
  • 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

    		1M238.3g/M22.40238.3g
    H2O mQ77.60825.7ml

    p=1.064

    HEPES, 1M, 1L

    Desired pHKOH, 5M
    [1000ml]
    5.250ml0ml
    5.350.5ml
    5.753.5ml
    6.037ml
    6.2412ml
    6.5922ml
    6.7132ml
    6.8845ml
    7.0050ml
    7.1060ml
    7.2580ml
    7.3792.5ml

    Imidazol

    C3H4N2, (store at 4oC):

    Conc.Stock50ml100ml
    Imidazole2M68.1g/M6.81g13.62g
    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)10ml50ml100ml150ml200ml
    PEG600040%37.214.0g20g40g60g80g
    H2OmQ62.796.75g33.75g67.5g101.25g135.0g

    p=1.075.

    PMSF

    (store at -20oC)