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        常用试剂配制

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        5005

        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.








          <center><font>About the recalculation of recipes for the arbitrary volume:<br /> <br /> </font> </center>

          TE

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

          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
          H2 O   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
          H2 O mQ   880µl 4.4ml
        • sterilize by autoclaving
        • STE

          1x; (store at 4o C).

          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
          H2 O   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
          H2 O 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
          Na3 C6 H5 O7 x 5.5H2 O 0.3M 357.1g/M 53.57g 160.7g 214.3g
          H2 O   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 4o C, 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
          ~
          ~
          H2 O   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

          H2 O     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 +4o C):

          Conc. Stock 0.5L 1.0L 2.0L
          MOPS (pH7.0) 1.0M 209.27g/M 104.64g 209.27g 418.54g
          AcONax3H2 O 0.5M 136.08g/M 34.02g 68.04g 136.08g
          EDTAx2H2 O 50mM 372.3g/M 9.31g 18.62g 37.23g
          H2 O   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 2H2 O 50mM 372.3g/M 9.31g 18.62g 37.23g
          H2 O   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 +4o C):

          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

          Na2 EDTAx2H2 O

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

          H2 O

            mQ 802ml 1203ml 1604ml 2005ml

          p=1.094

        • Taurine: C2 H7 NO3 S; Mw=125.15g/M.
        • TGB

          Tris-Glycine buffer 5x, pH8.3, (store the main stock at +4o C, 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
          H2 O     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

          H2 O   mQ 0.25ml 1.25ml 2.5ml 6.25ml

          PEG-NaCl

          (store at 4o C).

          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 4o C).

          Conc. Stock 10ml 50ml 100ml 150ml
          NaCl 0.1M 5M 200µl 1.0ml 2.0ml 3.0ml
          MgSO4~undefined 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
          H2 O   mQ 9.15ml 45.75ml 91.5ml 137.25ml

        • sterilize by autoclaving
        • STM

          (store at 4o C):

          Conc. Stock 50ml
          NaCl 10mM 5M 100µl
          Tris Cl pH8.0 50mM 1M 2.5ml
          MgCl2 10mM 1M 500µl
          H2 O   mQ 46.9ml

          TM

          (store at 4o C):

          Conc. Stock 50ml
          Tris Cl pH8.0 50mM 1M 2.5ml
          MgCl2 10mM 1M 500µl
          H2 O   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
          Na2 SO3 anhidr. 36.0g 57.6g 72.0g
          Hydroquinone 4.4g 7.04g 8.8g
          Na2 CO3 24.0g 38.4g 48.0g
          KBr 2.0g 3.2g 4.0g
          H2 O 490ml 784ml 980ml

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

          Fixer BKF-2

          (store at NT).

          500ml 800ml 1l
          Na2 S2 O3 x 5H2 O 130g 208g 260g
          NH4 Cl 25g 40g 50g
          Sodium pyrosulfite 8.5g 13.6g 17g
          H2 O 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 +4o C)

          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
          H2 O   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
          H2 O   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
          H2 O   mQ 73ml 127ml 190ml 317ml

          Mg2+ 2M

          (store at NT).

          Conc. Stock 200ml
          MgCl2 x6H2 O 1M 203.3g/M 40.66g
          MgSO4 x7H2 O 1M 246.48g/M 49.30g
          H2 O   mQ 144.6g

          p=1.173g/ml.

          Hybridization buffer HSB (f+ )

          high SDS hybridization buffer (store the main stock at 4o C, 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
          H2 O   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 H2 O+SSC+NaP).
        • Salmon sperm DNA should be shredded and denaturated.
        • Restriction buffers 10x

          А.

          Conc. Stock 100µl 1ml
          Tris Ac (pH7.9 37o C) 33mM 1M 33µl 330µl
          MgAc 10mM 1M 10µl 100µl
          KAc 66mM 5M 13.2µl 132µl
          DTT 0.5mM 1M 0.5µl 5µl
          H2 O   mQ 43.3µl 433µl

          B.

          Conc. Stock 100µl 1ml
          Tris Cl (pH 8.0 37o C)) 10mM 1M 10µl 100µl
          MgCl2 5mM 1M 5µl 50µl
          NaCl 100mM 5M 20µl 200µl
          b-Me 1mM 1M 1µl 10µl
          H2 O   mQ 64µl 640µl

          L.

          Conc. Stock 100µl 1ml
          Tris Cl (pH 7.5 37o C) 10mM 1M 10µl 100µl
          MgCl2 10mM 1M 10µl 100µl
          DTT 1mM 1M 1µl 10µl
          H2 O   mQ 79µl 790µl

          M.

          Conc. Stock 100µl 1ml
          Tris Cl (pH7.5 37o C) 10mM 1M 10µl 100µl
          NaCl 50mM 5M 10µl 100µl
          MgCl2 10mM 1M 10µl 100µl
          DTT 1mM 1M 1µl 10µl
          H2 O   mQ 69µl 690µl

          H.

          Conc. Stock 100µl 1ml
          Tris Cl (pH7.5 37o C) 50mM 1M 50µl 50µl
          MgCl2 10mM 1M 10µl 100µl
          NaCl 100mM 5M 20µl 200µl
          DTT 1mM 1M 1µl 10µl
          H2 0   mQ 19µl 190µl

          Denhardt solution 50x

          (store at -20o C)

          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
          H2 O   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 4o C):

          1x Conc. Stock 100ml 500ml
          KH2 PO4 1.7mM 17mM 136.1g/M 0.231g 1.157g
          Na2 HPO4 5.2mM 52mM 142g/M 0.738g 3.692g
          NaCl 150mM 1.5M 58.44g/M 8.77g 43.83g
          H2 O   mQ        
          1x Conc. Stock 100ml 500ml
          KH2 PO4 1.7mM 17mM 136.1g/M 0.231g 1.157g
          Na2 HPO4 x7H2 O 5.2mM 52mM 268.1g/M 1.394g 6.971g
          NaCl 150mM 1.5M 58.44g/M 8.77g 43.83g
          H2 O   mQ        

          PBS (recipe 2) 10x

          pH(1x)=7.3; (store at NT or at 4o C)
          1x 10x Stock 100ml 500ml
          KH2 PO4 1.47mM 14.7mM 136.1g/M 0.2g 1.0g
          Na2 HPO4 x7H2 O 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
          H2 O     mQ      

          PBS (recipe 3) 10x

          pH(1x)=7.3; (store at NT or at 4o C)
          1x 10x Stock 100ml 500ml
          KH2 PO4 2mM 20mM 136.1g/M 0.272g 1.36g
          Na2 HPO4 x7H2 O 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
          H2 O     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
          NaH2 PO4 xH2 O 200mM 137.99g/M 27.6g
          EDTA 20mM 372.24g/M 7.4g
          H2 O   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 -20o C, aliquote in use may be stored at 4o C

          RNAse storage buffer

          (store at -20o C).

          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

          H2 O   mQ 0.486g 2.43g 4.86g

          Proteinase K

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

          EDTA/glycogen solution

          20x (store at -20o C):

          Conc. Stock 1.0ml
          EDTA 0.2M 0.5M 400µl
          glycogen 1mg/ml 20mg/ml 50µl
          H2 O   mQ 550µl

          DNase RQ1 buffer

          10x (store at -20o C):

          Conc. Stock 1ml
          Tris Cl (pH8.025 o C ) 0.4M 1M 400µl
          NaCl 0.1M 5M 20µl
          MgCl2 60mM 1M 60µl
          CaCl2 0.1M 1M 100µl
          H2 0   mQ 420µl

          DNAse storage buffer

          (store at -20o C).

          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

          H2 O   mQ 0.485ml 0.73ml 1.21ml 2.42ml 4.85ml

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