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Compound solutions.

 

 
 
 
 
 
 
 
    About the recalculation of recipes for the arbitrary volume:

    TE

    1x; pH7.4; 7.6; 8.0; (store at 4oC).

    Conc.Stock1ml5ml10ml50ml400ml
    Tris Cl10mM1M10µl50µl100µl500µl4ml
    EDTA1mM0.5M2µl10µl20µl100µl800µl
    H2O mQ988µl4.94ml9.88ml49.4ml395ml

    10x:

    Conc.Stock1ml5ml
    Tris Cl0.1M1M0.1ml0.5ml
    EDTA10mM0.5M20µl0.1ml
    H2OmQ 880µl4.4ml
  • autoclave sterilization.
  • STE

    1x; (store at 4oC).

    Conc.Stock1ml5ml10ml50ml
    NaCl0.1M5M20µl100µl200µl1.0ml
    Tris Cl, pH8.010mM1M10µl50µl100µl0.5ml
    EDTA, pH8.01mM0.5M2µl10µl20µl100µl
    H2O mQ0.968ml4.84ml9.68ml48.4ml

    10x:

    Conc.Stock1ml5ml
    NaCl1.0M5M0.2ml1.0ml
    Tris Cl0.1M1M0.1ml0.5ml
    EDTA10mM0.5mM20µl0.1ml
    H2OmQ 680µl3.4ml
  • autoclave sterilization.
  • SSC

    20x; pH 7.0; (store at NT).

    Conc.Stock0.5L1.5L2L
    NaCl3M58.44g/M87.66g263.0g350.6g
    Na3C6H5O7x 5.5H2O0.3M357.1g/M53.57g160.7g214.3g
    H2O mQ440.3ml1.32L1.76L

    p =1.103

    TAE

    1x pH=7.6:

    Tris-acetate 40mM
    EDTA 1mM

    (store the large stock at 4oC, working quantity (~100ml) at NT)

    Conc.Stock100ml150ml200ml250ml
    Tris-base2M121.1g/M24.22g36.33g48.44g60.55g
    EDTA0.05M372.3g/M1.862g2.792g3.723g4.654g
    Acetic acid~1M17.4M ~5.71ml
    ~6.00g
    ~8.57ml
    ~9.00g
    ~11.42ml
    ~12.00g
    ~14.28ml
    ~15.00g

    H2O mQ     

    p(50x)=1.0878

  • it is convenient to solubilize Tris and EDTA first, and only then add acid to adjust pH.
  • TBE

    10x, pH ~8.3;(store at NT in clean bottle)

    1x10xStock1L
    Tris base89mM0.89M121.14g/M107.8g
    Boric acid89mM0.89M61.83g/M55.03g
    EDTA, pH 8.02mM20mM500mM40ml
    43.84g

    H2O  mQ863.3ml
  • 10x stock solution is stable at NT if to clean it through 0.4µm filter;
  • for PAAG it is used as 1x, for agarose gel - as 0.5x;
  • for agarose gels it is used for separation of small (< 200bp) fragments. Generally accepted, that the purification of fragments is more complex from TBE gels.
  • FGRB

    50x (formaldehyde gel-running buffer), p=1.104g/ml (store at +4oC):

    Conc.Stock0.5L1.0L2.0L
    MOPS (pH7.0)1.0M209.27g/M104.64g209.27g418.54g
    AcONax3H2O0.5M136.08g/M34.02g68.04g136.08g
    EDTAx2H2O50mM372.3g/M9.31g18.62g37.23g
    H2O mQ404.0ml808ml1616ml
    Conc.Stock0.5L1.0L2.0L
    MOPS (pH7.0)1.0M209.27g/M104.64g209.27g418.54g
    AcONa anhydrous0.5M82.04g/M20.51g41.02g82.04g
    EDTA x 2H2O50mM372.3g/M9.31g18.62g37.23g
    H2O mQ417.5ml835ml1670ml
  • adjust pH7.0 10N NaOH (per 500ml of buffer ~22ml), filter-sterilize through 0.22µm filter;
  • solution becomes yellow during the storage on the light, but it has no effect on the solution quality.
  • GTGB

    20x /Glycerol tolerant gel buffer/ (store at +4oC):

    Conc.Stock1L1.5L2.0L2.5L

    Tris base

    1.78M121.14g/M216g324g432g540g

    Taurine

    0.56M125.15g/M72g108g144g180g

    Na2EDTAx2H2O

    10.7mM372.3 g/M4g6g8g10g

    H2O

     mQ802ml1203ml1604ml2005ml

    p=1.094

  • Taurine: C2H7NO3S; Mw=125.15g/M.
  • TGB

    Tris-Glycine buffer 5x, pH8.3, (store the main stock at +4oC, working quantity at NT).

    1xConc.Stock1L1.5L
    Tris-base25mM125mM121.1g/M15.1g22.71g
    Glycine250mM1.25M75.05g/M94g141g
    SDS0.1%0.5%10%50ml75ml
    H2O  mQ   

    Loading buffer

    10x, (store at NT).

    Conc.Stock1ml5ml10ml25ml
    Xilene cyanol0.1%solid1.0mg5.0mg10mg25.0mg
    Bromph. blue0.1%solid1.0mg5.0mg10mg25.0mg
    SDS0.5%10%50µl250µl500µl1.25ml
    EDTA, pH8.00.1M0.5M0.2ml1.0ml2.0ml5.0ml
    Glycerol50%100% 0.5ml
    0.625g
    2.5ml
    3.125g
    5.0ml
    6.25g
    12.5ml
    15.63g

    H2O mQ0.25ml1.25ml2.5ml6.25ml

    PEG-NaCl

    (store at 4oC).

    Conc.Stock50ml100ml150ml200ml
    NaCl1.6M58.44g/M4.675g9.35g14.03g18.70g
    PEG-600013%solid6.50g13.00g19.50g26.00g

    or:

    Conc.Stock50ml100ml150ml200ml
    NaCl1.6M5M 16ml
    18.97g
    32ml
    37.95g
    48ml
    56.92g
    64ml
    75.90g

    PEG-600013%40%16.25ml32.50ml48.75ml65.00ml

    SM

    (store at 4oC).

    Conc.Stock10ml50ml100ml150ml
    NaCl0.1M5M200µl1.0ml2.0ml3.0ml
    MgSO4*10mM1M100µl0.5ml1.0ml1.5ml
    Tris Cl, pH7.550mM1M0.5ml2.5ml5.0ml7.5ml
    Gelatin0.01%2%50µl0.25ml0.5ml0.75ml
    H2O mQ9.15ml45.75ml91.5ml137.25ml

    * - add after autoclaving;

  • autoclave sterilization.
  • STM

    (store at 4oC):

    Conc.Stock50ml
    NaCl10mM5M100µl
    Tris Cl pH8.050mM1M2.5ml
    MgCl210mM1M500µl
    H2O mQ46.9ml

    TM

    (store at 4oC):

    Conc.Stock50ml
    Tris Cl pH8.050mM1M2.5ml
    MgCl210mM1M500µl
    H2O mQ47.0ml

    Denaturation buffer

    (store at NT).

    Conc.Stock100ml150ml300ml500ml750ml
    NaCl1.5M58.44g/M8.766g13.15g26.30g43.83g65.75g
    NaOH0.5M10M 5ml
    6.65g
    7.5ml
    9.97g
    15ml
    19.94g
    25ml
    33.23g
    37.5ml
    49.84g

      40g/M2g3g6g10g15g

    Neutralization buffer

    (store at NT).

    Conc.Stock150ml300ml500ml750ml
    Tris Cl, pH7.41M121.1g/M18.15g36.3g60.5g90.75g
    NaCl1.5M58.44g/M13.15g26.30g43.83g65.75g
    HCl~0.81M11.6M/L~10.5ml~21ml~35ml~52.5ml
    EDTA (optional)1mM0.5M0.3ml0.6ml1ml1.5ml

    Developer Rentgen 2

    (store at NT in the dark).

    500ml800ml1l
    Methol1.1g1.76g2.2g
    Na2SO336.0g57.6g72.0g
    Hydroquinone4.4g7.04g8.8g
    Na2CO324.0g38.4g48.0g
    KBr2.0g3.2g4.0g
    H2O490ml784ml980ml

    solubilize in the presented order; r=1.115.

    Fixer BKF 2

    (store at NT).

    500ml800ml1l
    Na2S2O3x 5H2O130g208g260g
    NH4Cl25g40g50g
    Sodium pyrosulfite8.5g13.6g17g
    H2O398ml636.8ml796ml

    solubilize in the presented order; r=1.123.

    Scintillation solution

    (store at NT in the dark):

    0.5L1L
    PPO2.50g5.00g
    POPOP0.05g0.10g
    Toluene   

    I plasmid solution

    (store at +4oC)

    Conc.Stock50ml100ml150ml200ml
    Glucose50mM2M 1.25ml
    1.41g
    2.5ml
    2.825g
    3.75ml
    4.24g
    5.0ml
    5.56g

    Tris Cl, pH 8.025mM1.0 M1.25ml2.5ml3.75ml5.0ml
    EDTA10mM0.5 M1.0ml2.0ml3.0ml4.0ml
    H2O mQ46.5ml93ml139.5ml186ml

    II plasmid solution

    Conc.Stock0.5ml1ml2.5ml5.0ml6.5ml8.5ml11ml
    NaOH0.2N1N0.1ml0.2ml0.5ml1.0ml1.3ml1.7ml2.2ml
    SDS1%10%0.05ml0.1ml0.25ml0.5ml0.65ml0.85ml1.1ml
    H2O mQ0.350ml0.7ml1.75ml3.5ml4.55ml5. 95ml7.7ml

    Oligonucleotide gel staining mix

    (store at NT).

    Conc.Stock100ml200ml300ml500ml
    AcONa(5.2)0.5M3M17ml33ml50.0ml83ml
    Met. blue0.04%0.2%20ml40ml60ml100ml
    H2O mQ73ml127ml190ml317ml

    Mg2+2M

    (store at NT).

    Conc.Stock200ml
    MgCl2x6H2O1M203.3g/M40.66g
    MgSO4x7H2O1M246.48g/M49.30g
    H2O mQ144.6g

    p=1.173g/ml.

    HSB (f+)

    high SDS hybridization buffer (store the main stock at 4oC, working - NT)

    Conc.Stock500ml
    SDS7%solid35g
    Formamid50%100%250ml
    283.3g

    SSC5x20x125ml
    145.3g

    Bloc. Reagent2%solid10g
    NaP (pH 7.0)50mM1M25ml
    27.6g

    N-Lauroylsarcosine Na0.1%10%5ml
    5.1g

    Salm.Sperm DNA50µg/ml10µg/l2.5ml
    H2O mQ49ml

    p=1.111

  • Blocking Reagent - is the powder from "Roche Diagnostics GmBH; former Boehringer Mannheim" #1096176. Instead of 2% Blocking Reagent one may use 10x Denhardt or 2% non-fat dry milk.
  • it is possible to dissolve the Blocking Reagent only in high strength buffer (in this particular case in H2O+SSC+NaP).
  • Salmon sperm DNA should be shredded and denaturated.
  • Restriction buffers 10x

    .

    Conc.Stock100µl1ml
    Tris Ac (pH7.9 37oC)33mM1M33µl330µl
    MgAc10mM1M10µl100µl
    KAc66mM5M13.2µl132µl
    DTT0.5mM1M0.5µl5µl
    H2O mQ43.3µl433µl

    B.

    Conc.Stock100µl1ml
    Tris Cl (pH 8.0 37oC))10mM1M10µl100µl
    MgCl25mM1M5µl50µl
    NaCl100mM5M20µl200µl
    b-MeEtOH1mM1M1µl10µl
    H2O mQ64µl640µl

    L.

    Conc.Stock100µl1ml
    Tris Cl (pH 7.5 37oC)10mM1M10µl100µl
    MgCl210mM1M10µl100µl
    DTT1mM1M1µl10µl
    H2O mQ79µl790µl

    M.

    Conc.Stock100µl1ml
    Tris Cl (pH7.5 37oC)10mM1M10µl100µl
    NaCl50mM5M10µl100µl
    MgCl210mM1M10µl100µl
    DTT1mM1M1µl10µl
    H2O mQ69µl690µl

    H.

    Conc.Stock100µl1ml
    Tris Cl (pH7.5 37oC)50mM1M50µl50µl
    MgCl210mM1M10µl100µl
    NaCl100mM5M20µl200µl
    DTT1mM1M1µl10µl
    H20 mQ19µl190µl

    Denhardt solution 50x

    (store at -20oC)

    Conc.Stock50ml300ml1L
    Ficoll 4001%solid0.5g3.0g10g
    Polyvinilpyrolidone1%solid0.5g3.0g10g
    BSA (fr V)1%solid0.5g3.0g10g
    H2O mQ46.7ml292.2ml974ml

    p=1.004

  • filter purification;
  • it is possible to prepare 100x.
  • PBS

    pH(1x)=7.4, (store at NT or at 4oC):

    1xConc.Stock100ml500ml
    KH2PO41.7mM17mM136.1g/M0.231g1.157g
    Na2HPO45.2mM52mM142g/M0.738g3.692g
    NaCl150mM1.5M58.44g/M8.77g43.83g
    H2O mQ    
    1xConc.Stock100ml500ml
    KH2PO41.7mM17mM136.1g/M0.231g1.157g
    Na2HPO4x7H2O5.2mM52mM268.1g/M1.394g6.971g
    NaCl150mM1.5M58.44g/M8.77g43.83g
    H2O mQ    
  • autoclave sterilization;
  • pH is concentration dependent; thus check pH only for diluted 1x buffer;
  • pH of 10x should be ~6.8 (adjust by NaOH if necessary).
  • SSPE

    pH 7.4

    Conc.Stock1L
    NaCl3M58.44g/M175g
    NaH2PO4xH2O200mM137.99g/M27.6g
    EDTA20mM372.24g/M7.4g
  • adjust pH by 10N NaOH.
  • RNase A (DNase free)

    (store at -20oC, working quantities may be stored at 4oC)

    1. solubilize 10mg/ml in 1-50mM AcONa (pH4.5);
    2. 15' in boiling water bath, then cool slowly to NT;

    RNAse storage buffer

    (store at -20oC).

    Conc.Stock1ml5ml10ml
    Tris Cl, pH7.610mM1M10µl50µl100µl
    Glycerol50%100% 0.5ml
    0.630g
    2.5ml
    3.152g
    5.0ml
    6.305g

    H2O mQ0.486g2.43g4.86g

    Proteinase K

    (store at +4oC, long-term storage at 20oC):
    20mg/ml solution in 10mM Tris Cl, 1.0mM CaCl2, 30% glycerol.

    EDTA/glycogen

    20x (store at -20oC):

    Conc.Stock1.0ml
    EDTA0.2M0.5M400 µl
    glycogen1mg/ml20mg/ml50 µl
    H2O mQ550 µl

    DNase RQ1 buffer

    10x (store at -20oC):

    Conc.Stock1ml
    Tris Cl (pH8.025oC)0.4M1M400µl
    NaCl0.1M5M20µl
    MgCl260mM1M60µl
    CaCl20.1M1M100µl
    H20 mQ420µl

    DNAse storage buffer

    (store at -20oC).

    Conc.Stock1ml1.5ml2.5ml5ml10ml
    Tris Cl, pH 7.020mM1M20µl30µl50µl100µl200µl
    NaCl50mM5M10µl15µl25µl50µl100µl
    DTT1mM1M1µl1.5µl2.5µl5.0µl10µl
    Glycerol50%100% 0.5ml
    0.630g
    0.75ml
    0.9g
    1.25ml
    1.58g
    2.5ml
    3.15g
    5.0ml
    6.31g

    H2O mQ0.485g0.73g1.21g2.42g4.85g

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