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常用试剂配制

信息来源：本站原创　更新时间：2004-7-26 13:51:00
TE 1x, 10x
STE 1x, 10x
SSC 20x
Electrophoresis buffers.
TAE 50x
TBE 10x
FGRB 50x
GTGB 20x
TGB 5x
Loading buffer 10x
PEG-NaCl
Bacteriophage lambda buffers
SM
STM
TM
Denaturation buffer
Neutraliszation buffer
Developer Rentgen-2
Fixer BKF-2
Scintillation solution
Plasmid solution I
Plasmid solution II
Oligonucleotide gel staining mix
2M Mg2+ solution
Hybridization buffer HSB (f+)
Restriction buffers
А., B., L., M., H.
Denhardt solution 50х
PBS 10x
SSPE 20x
RNase (DNase free)

Proteinase K solution
EDTA/glycogen solution 20x
DNase RQ1 buffer

 Supplement.








    About the recalculation of recipes for the arbitrary volume:

    TE

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

    Conc. Stock 1ml 5ml 10ml 50ml 400ml
    Tris Cl 10mM 1M 10µl 50µl 100µl 500µl 4ml
    EDTA 1mM 0.5M 2µl 10µl 20µl 100µl 800µl
    H2O   mQ 988µl 4.94ml 9.88ml 49.4ml 395ml

    10x:

    Conc. Stock 1ml 5ml
    Tris Cl 0.1M 1M 0.1ml 0.5ml
    EDTA 10mM 0.5M 20µl 0.1ml
    H2O mQ   880µl 4.4ml
  • sterilize by autoclaving

    • STE

    1x; (store at 4oC).

    Conc. Stock 1ml 5ml 10ml 50ml
    NaCl 0.1M 5M 20µl 100µl 200µl 1.0ml
    Tris Cl, pH8.0 10mM 1M 10µl 50µl 100µl 0.5ml
    EDTA, pH8.0 1mM 0.5M 2µl 10µl 20µl 100µl
    H2O   mQ 0.968ml 4.84ml 9.68ml 48.4ml

    10x:

    Conc. Stock 1ml 5ml
    NaCl 1.0M 5M 0.2ml 1.0ml
    Tris Cl 0.1M 1M 0.1ml 0.5ml
    EDTA 10mM 0.5mM 20µl 0.1ml
    H2O mQ   680µl 3.4ml
  • sterilize by autoclaving

    • SSC

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

    Conc. Stock 0.5L 1.5L 2L
    NaCl 3M 58.44g/M 87.66g 263.0g 350.6g
    Na3C6H5O7x 5.5H2O 0.3M 357.1g/M 53.57g 160.7g 214.3g
    H2O   mQ 440.3ml 1.32L 1.76L

    p = 1.103

    TAE

    1x pH = 7.6:

    Tris-acetate 40mM
    EDTA 1mM

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

    Conc. Stock 100ml 150ml 200ml 250ml
    Tris-base 2M 121.1g/M 24.22g 36.33g 48.44g 60.55g
    EDTA 0.05M 372.3g/M 1.862g 2.792g 3.723g 4.654g
    Acetic acid ~1.56M 17.4M ~8.96ml
    ~9.40g    		 ~13.44ml
    ~14.10g    		 ~17.92ml
    ~18.80g    		 ~22.40ml
    ~23.50g    		 ~
    ~
    H2O   mQ ~73.30g ~109.95g ~146.60g ~183.25g ~

    p(50x)=1.0878

    it is convenient to solubilize Tris and EDTA first then adjust pH by acetic acid.

    TBE

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

    1x 10x Stock 1L
    Tris base 89mM 0.89M 121.14g/M 107.8g
    Boric acid 89mM 0.89M 61.83g/M 55.03g
    EDTA, pH 8.0 2mM 20mM 500mM 40ml
    43.84g
    H2O     mQ 863.3ml
  • 10x stock solution is stable at NT after 0.4µm filtration;
  • for PAAG use as 1x, for agarose gel - as 0.5x;
  • TBE is used in agarose gels for separation of small (< 200bp) fragments. Purification of fragments from TBE-based gels is more complicated than from TAE-based gels.

    • FGRB

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

    Conc. Stock 0.5L 1.0L 2.0L
    MOPS (pH7.0) 1.0M 209.27g/M 104.64g 209.27g 418.54g
    AcONax3H2O 0.5M 136.08g/M 34.02g 68.04g 136.08g
    EDTAx2H2O 50mM 372.3g/M 9.31g 18.62g 37.23g
    H2O   mQ 404.0ml 808ml 1616ml
    Conc. Stock 0.5L 1.0L 2.0L
    MOPS (pH7.0) 1.0M 209.27g/M 104.64g 209.27g 418.54g
    AcONa anhydrous 0.5M 82.04g/M 20.51g 41.02g 82.04g
    EDTA x 2H2O 50mM 372.3g/M 9.31g 18.62g 37.23g
    H2O   mQ 417.5ml 835ml 1670ml
  • 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. Stock 1L 1.5L 2.0L 2.5L

    Tris base

    		 1.78M 121.14g/M 216g 324g 432g 540g

    Taurine

    		 0.56M 125.15g/M 72g 108g 144g 180g

    Na2EDTAx2H2O

    		 10.7mM 372.3 g/M 4g 6g 8g 10g

    H2O

    		   mQ 802ml 1203ml 1604ml 2005ml

    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).

    1x Conc. Stock 1L 1.5L
    Tris-base 25mM 125mM 121.1g/M 15.1g 22.71g
    Glycine 250mM 1.25M 75.05g/M 94g 141g
    SDS 0.1% 0.5% 10% 50ml 75ml
    H2O     mQ      

    Loading buffer

    10x, (store at NT).

    Conc. Stock 1ml 5ml 10ml 25ml
    Xilene cyanol 0.1% solid 1.0mg 5.0mg 10mg 25.0mg
    Bromph. blue 0.1% solid 1.0mg 5.0mg 10mg 25.0mg
    SDS 0.5% 10% 50µl 250µl 500µl 1.25ml
    EDTA, pH8.0 0.1M 0.5M 0.2ml 1.0ml 2.0ml 5.0ml
    Glycerol 50% 100% 0.5ml
    0.625g    		 2.5ml
    3.125g    		 5.0ml
    6.25g    		 12.5ml
    15.63g
    H2O   mQ 0.25ml 1.25ml 2.5ml 6.25ml

    PEG-NaCl

    (store at 4oC).

    Conc. Stock 50ml 100ml 150ml 200ml
    NaCl 1.6M 58.44g/M 4.675g 9.35g 14.03g 18.70g
    PEG-6000 13% solid 6.50g 13.00g 19.50g 26.00g

    or:

    Conc. Stock 50ml 100ml 150ml 200ml
    NaCl 1.6M 5M 16ml
    18.97g    		 32ml
    37.95g    		 48ml
    56.92g    		 64ml
    75.90g
    PEG-6000 13% 40% 16.25ml 32.50ml 48.75ml 65.00ml

    SM

    (store at 4oC).

    Conc. Stock 10ml 50ml 100ml 150ml
    NaCl 0.1M 5M 200µl 1.0ml 2.0ml 3.0ml
    MgSO4* 10mM 1M 100µl 0.5ml 1.0ml 1.5ml
    Tris Cl, pH7.5 50mM 1M 0.5ml 2.5ml 5.0ml 7.5ml
    0.01% 2% 50µl 0.25ml 0.5ml 0.75ml
    H2O   mQ 9.15ml 45.75ml 91.5ml 137.25ml

    * - add after autoclaving;

  • sterilize by autoclaving

    • STM

    (store at 4oC):

    Conc. Stock 50ml
    NaCl 10mM 5M 100µl
    Tris Cl pH8.0 50mM 1M 2.5ml
    MgCl2 10mM 1M 500µl
    H2O   mQ 46.9ml

    TM

    (store at 4oC):

    Conc. Stock 50ml
    Tris Cl pH8.0 50mM 1M 2.5ml
    MgCl2 10mM 1M 500µl
    H2O   mQ 47.0ml

    Denaturation buffer

    (store at NT).

    Conc. Stock 100ml 150ml 300ml 500ml 750ml
    NaCl 1.5M 58.44g/M 8.766g 13.15g 26.30g 43.83g 65.75g
    NaOH 0.5M 10M 5ml
    6.65g    		 7.5ml
    9.97g    		 15ml
    19.94g    		 25ml
    33.23g    		 37.5ml
    49.84g
        40g/M 2g 3g 6g 10g 15g

    Neutralization buffer

    (store at NT).

    Conc. Stock 150ml 300ml 500ml 750ml
    Tris Cl, pH7.4 1M 121.1g/M 18.15g 36.3g 60.5g 90.75g
    NaCl 1.5M 58.44g/M 13.15g 26.30g 43.83g 65.75g
    HCl ~0.81M 11.6M/L ~10.5ml ~21ml ~35ml ~52.5ml
    EDTA (optional) 1mM 0.5M 0.3ml 0.6ml 1ml 1.5ml

    Developer Rentgen-2

    (store at NT in the dark).

    500ml 800ml 1l
    1.1g 1.76g 2.2g
    Na2SO3 anhidr. 36.0g 57.6g 72.0g
    Hydroquinone 4.4g 7.04g 8.8g
    Na2CO3 24.0g 38.4g 48.0g
    KBr 2.0g 3.2g 4.0g
    H2O 490ml 784ml 980ml

    solubilize in the presented order , p=1.115g/ml.

    Fixer BKF-2

    (store at NT).

    500ml 800ml 1l
    Na2S2O3x 5H2O 130g 208g 260g
    NH4Cl 25g 40g 50g
    Sodium pyrosulfite 8.5g 13.6g 17g
    H2O 398ml 636.8ml 796ml

    solubilize in the presented order, p=1.123g/ml.

    Scintillation solution

    (store at NT in the dark):

    0.5L 1L
    PPO 2.50g 5.00g
    POPOP 0.05g 0.10g
    Toluene      

    Plasmid solution I

    (store at +4oC)

    Conc. Stock 50ml 100ml 150ml 200ml
    Glucose 50mM 2M 1.25ml
    1.41g    		 2.5ml
    2.825g    		 3.75ml
    4.24g    		 5.0ml
    5.56g
    Tris Cl, pH 8.0 25mM 1.0 M 1.25ml 2.5ml 3.75ml 5.0ml
    EDTA 10mM 0.5 M 1.0ml 2.0ml 3.0ml 4.0ml
    H2O   mQ 46.5ml 93ml 139.5ml 186ml

    Plasmid solution II

    Conc. Stock 0.5ml 1ml 2.5ml 5.0ml 6.5ml 8.5ml
    NaOH 0.2N 1N 0.1ml 0.2ml 0.5ml 1.0ml 1.3ml 1.7ml
    SDS 1% 10% 0.05ml 0.1ml 0.25ml 0.5ml 0.65ml 0.85ml
    H2O   mQ 0.350ml 0.7ml 1.75ml 3.5ml 4.55ml 5.95ml

    Oligonucleotide gel staining mix

    (store at NT).

    Conc. Stock 100ml 200ml 300ml 500ml
    AcONa(5.2) 0.5M 3M 17ml 33ml 50.0ml 83ml
    Met. blue 0.04% 0.2% 20ml 40ml 60ml 100ml
    H2O   mQ 73ml 127ml 190ml 317ml

    Mg2+2M

    (store at NT).

    Conc. Stock 200ml
    MgCl2x6H2O 1M 203.3g/M 40.66g
    MgSO4x7H2O 1M 246.48g/M 49.30g
    H2O   mQ 144.6g

    p=1.173g/ml.

    Hybridization buffer HSB (f+)

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

    Conc. Stock 500ml
    SDS 7% solid 35g
    Formamid 50% 100% 250ml
    283.3g
    SSC 5x 20x 125ml
    145.3g
    Bloc. Reagent 2% solid 10g
    NaP (pH 7.0) 50mM 1M 25ml
    27.6g
    N-Lauroylsarcosine Na 0.1% 10% 5ml
    5.1g
    Salm.Sperm DNA 50µg/ml 10µg/µl 2.5ml
    H2O   mQ 49ml

    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

    А.

    Conc. Stock 100µl 1ml
    Tris Ac (pH7.9 37oC) 33mM 1M 33µl 330µl
    MgAc 10mM 1M 10µl 100µl
    KAc 66mM 5M 13.2µl 132µl
    DTT 0.5mM 1M 0.5µl 5µl
    H2O   mQ 43.3µl 433µl

    B.

    Conc. Stock 100µl 1ml
    Tris Cl (pH 8.0 37oC)) 10mM 1M 10µl 100µl
    MgCl2 5mM 1M 5µl 50µl
    NaCl 100mM 5M 20µl 200µl
    b-Me 1mM 1M 1µl 10µl
    H2O   mQ 64µl 640µl

    L.

    Conc. Stock 100µl 1ml
    Tris Cl (pH 7.5 37oC) 10mM 1M 10µl 100µl
    MgCl2 10mM 1M 10µl 100µl
    DTT 1mM 1M 1µl 10µl
    H2O   mQ 79µl 790µl

    M.

    Conc. Stock 100µl 1ml
    Tris Cl (pH7.5 37oC) 10mM 1M 10µl 100µl
    NaCl 50mM 5M 10µl 100µl
    MgCl2 10mM 1M 10µl 100µl
    DTT 1mM 1M 1µl 10µl
    H2O   mQ 69µl 690µl

    H.

    Conc. Stock 100µl 1ml
    Tris Cl (pH7.5 37oC) 50mM 1M 50µl 50µl
    MgCl2 10mM 1M 10µl 100µl
    NaCl 100mM 5M 20µl 200µl
    DTT 1mM 1M 1µl 10µl
    H20   mQ 19µl 190µl

    Denhardt solution 50x

    (store at -20oC)

    Conc. Stock 50ml 300ml 1L
    Ficoll 400 1%(w/v) solid 0.5g 3.0g 10g
    Polyvinilpyrolidone 1%(w/v) solid 0.5g 3.0g 10g
    BSA (fr V) 1%(w/v) solid 0.5g 3.0g 10g
    H2O   mQ 46.7ml 292.2ml 974ml

    p=1.004

  • the solution should be filtered;
  • the solution could be prepared as 100x stock.

    • PBS (recipe 1)

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

    1x Conc. Stock 100ml 500ml
    KH2PO4 1.7mM 17mM 136.1g/M 0.231g 1.157g
    Na2HPO4 5.2mM 52mM 142g/M 0.738g 3.692g
    NaCl 150mM 1.5M 58.44g/M 8.77g 43.83g
    H2O   mQ        
    1x Conc. Stock 100ml 500ml
    KH2PO4 1.7mM 17mM 136.1g/M 0.231g 1.157g
    Na2HPO4x7H2O 5.2mM 52mM 268.1g/M 1.394g 6.971g
    NaCl 150mM 1.5M 58.44g/M 8.77g 43.83g
    H2O   mQ        

    PBS (recipe 2) 10x

    pH(1x)=7.3; (store at NT or at 4oC)
    1x 10x Stock 100ml 500ml
    KH2PO4 1.47mM 14.7mM 136.1g/M 0.2g 1.0g
    Na2HPO4x7H2O 4.29mM 42.9mM 268.1g/M 1.15g 5.75g
    NaCl 137mM 1.37M 58.44g/M 8.0g 40.0g
    KCl 2.68mM 26.8mM 74.56g/M 0.2g 1.0g
    H2O     mQ      

    PBS (recipe 3) 10x

    pH(1x)=7.3; (store at NT or at 4oC)
    1x 10x Stock 100ml 500ml
    KH2PO4 2mM 20mM 136.1g/M 0.272g 1.36g
    Na2HPO4x7H2O 10mM 100mM 268.1g/M 2.68g 13.41g
    NaCl 137mM 1.37M 58.44g/M 8.0g 40.0g
    KCl 2.7mM 27mM 74.56g/M 0.20g 1.01g
    H2O     mQ      
  • sterilize by autoclaving;
  • pH is concentration dependent; therefore check pH for diluted 1x buffer only;
  • pH of 10x should be ~6.8 (adjust by NaOH if necessary).

    • SSPE 20x

    pH 7.4

    Conc. Stock 1L
    NaCl 3M 58.44g/M 175g
    NaH2PO4xH2O 200mM 137.99g/M 27.6g
    EDTA 20mM 372.24g/M 7.4g
    H2O   mQ 926ml
  • adjust pH by 10N NaOH.

    • RNase A (DNase free)

    1. solubilize 10mg/ml in 1-50mM AcONa (pH4.5);
    2. incubate 15' in boiling water bath, then cool slowly to NT;
    3. store at -20oC, aliquote in use may be stored at 4oC

    RNAse storage buffer

    (store at -20oC).

    Conc. Stock 1ml 5ml 10ml
    Tris Cl, pH7.6 10mM 1M 10µl 50µl 100µl
    Glycerol 50% 100% 0.5ml
    0.630g    		 2.5ml
    3.152g    		 5.0ml
    6.305g
    H2O   mQ 0.486g 2.43g 4.86g

    Proteinase K

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

    EDTA/glycogen solution

    20x (store at -20oC):

    Conc. Stock 1.0ml
    EDTA 0.2M 0.5M 400µl
    glycogen 1mg/ml 20mg/ml 50µl
    H2O   mQ 550µl

    DNase RQ1 buffer

    10x (store at -20oC):

    Conc. Stock 1ml
    Tris Cl (pH8.025oC) 0.4M 1M 400µl
    NaCl 0.1M 5M 20µl
    MgCl2 60mM 1M 60µl
    CaCl2 0.1M 1M 100µl
    H20   mQ 420µl

    DNAse storage buffer

    (store at -20oC).

    Conc. Stock 1ml 1.5ml 2.5ml 5ml 10ml
    Tris Cl, pH 7.0 20mM 1M 20µl 30µl 50µl 100µl 200µl
    NaCl 50mM 5M 10µl 15µl 25µl 50µl 100µl
    DTT 1mM 1M 1µl 1.5µl 2.5µl 5.0µl 10µl
    Glycerol 50% 100% 0.5ml
    0.630g    		 0.75ml
    0.9g    		 1.25ml
    1.58g    		 2.5ml
    3.15g    		 5.0ml
    6.31g
    H2O   mQ 0.485ml 0.73ml 1.21ml 2.42ml 4.85ml
实验频道录入：Protocol    责任编辑：Protocol 

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