طرز تهیه انواع بافرها و محلول های آزمایشگاه مولکولی

SOLUTION RECIPES  DYES, STAINS, AND COLORANTS 6X Agarose Gel Loading Dye                                                                                     200 mL             Glycerol                                                          30 mL             Bromophenol Blue                                       0.5 g             Xylene Cyanol                                                0.5 g Destain                                                                                     1 L             Methanol                                                      50 mL             Acetic Acid                                                     42 mL              1 mg/mL Ethidium Bromide                                                                                     50 mL             Ethidium Bromide                                       50 mg             Wrap bottle in aluminum foil. 5X Ficoll/Loading Dye (Orange Dye)                                                                         25 mL Ficoll                                                               6.75 g              Orange G                                                        0.25 g              Heat to dissolve (use 50 mL conical in water bath).  Aliquot and store at -20°C. Formamide Loading Buffer                                                                             10 mL Formamide                                                      9.5 mL Bromophenol Blue                                        0.009 mg Xylene Cyanol FF                                           0.009 mg Store at 4°C HTA Loading Buffer                                                                                                            10 mL             Tris                                                                  0.03g             Glycerol                                                          5 mL             1 M DTT                                                         5 μL             Bromophenol Blue                                      pinch             Xylene Cyanol                                              pinch SDS Lysis Buffer                                                                                     50 mL             SDS                                                                 0.5 g             Glycerol                                                          5 mL             β-Mercapto Ethanol                                     5 mL             1M Tris, pH 6.8                                              2 mL             Bromophenol Blue                                        pinch             Xylene Cyanol Stain                                                                                                   1 L             Methanol                                                       150 mL             Acetic Acid                                                     100 mL             Coomassie Blue                                              2 g Urea Loading Buffer                                                                                     10 mL             Urea                                                                4.8 g             EDTA                                                             0.074 g             1 M Tris pH 7.5                                               50 μL             Xylene Cyanol                                                0.025 g             Bromophenol Blue                                       0.025 g BUFFERS 10X Annealing Buffer                                                                                     10 mL             1 M Tris-HCl pH 7.5                                       1 mL             2 M KCl                                                          1.25 mL Blot Transfer Buffer                                                                                       1 L             Methanol                                                       200 mL             Glycine                                                            14.4 g             Tris                                                                  3.03 g Breaking Buffer                                                                       10 mL 10% Triton X-100                                        2 mL                            10% SDS                                                        1 mL    5M NaCl                                                         200 ml              1M Tris-Cl, pH8.0                                        100 ml              0.5M EDTA                                                    20 ml                            Sigma water                                                 6.68 mL           Carbonate Buffer                                                                                     1 L             Sodium Carbonate                                      8.4 g             Magnesium Chloride                                  0.203 g             pH to 9.8 Cell Competency Buffer                                                                          500mL 100% Glycerol                                                70 mL Calcium Chloride                                            50 mL Denaturing Buffer                                                                                    10 mL             Deionized Formamide                                 5 mL             Formalin                                                        2 mL             10X MOPS                                                     1 mL             DEPC H2O                                                      2 mL Hirt Lysis Buffer                                                                                     100 mL             10% SDS                                                          6 mL             1 M Tris-HCl, pH 7.4                                      1 mL             1 M EDTA, pH 8.0                                          1 mL Hybridization Buffer                                                                                     500 mL             10% SDS                                                           5 mL             Liquid Blocking Agent                                   25 mL             5X SSC                                                              470 mL             Make 10 mL aliquots and store in –20 freezer 4X Lower Tris                                                                                     1 L             Tris                                                                  181.71 g             PH to 8.8 10X MOPS Buffer                                                                                     1 L             MOPS                                                             83.72 g             Sodium Acetate                                              13.61 g             0.5M EDTA                                                      20 mL             pH to 7.0             Autoclave NETS Buffer                                                                                     500 mL                        1M NaCl                                                         50 mL             100mM Tris HCl, pH 7.6                              50 mL             0.5M EDTA                                                    1 mL             10 % SDS                                                       25 mL 10X Non-Denaturing Gel Shift Buffer                                                                             1 L Tris                                                                   30.28 g Glycine                                                            144.1 g EDTA                                                                3.72 g Filter sterilize 10X PBS                                                                                     500 mL             NaCl                                                                40 g             KCl                                                                   1 g             Na2PO4                                                            7.2 g             KH2PO4                                                            1.2 g             pH to 7.5 1X PBS – FA                                                                                      1 L             NaCl                                                                8.5 g             NaH2PO4 – H2O                                             0.14 g             Na2HPO4                                                                       1.28 g             pH to 7.5 10X PCR Buffer                                                                                     100 mL             KCl                                                                 3.728 g             1M Tris-HCl, pH 9.0                                    10 mL             MgCl2                                                                                               0.143 g             Triton-X 100                                                  1 mL Polynucleotide Kinase Storage Buffer                                                                                     50 mL             1M Tris-HCl                                                    2.5 mL             10 mM EDTA                                                 500 ml             1 M DTT                                                         50 ml             Glycerol                                                          25 mL Renaturation Buffer                                                                         100 mL                                    1M Tris HCl, pH 7.5                                       1 mL NaCl                                                                  0.146 g 1M MgCl2                                                                                        200 μL 5X RT Buffer                                                                                     10 mL             Tris-HCl, pH 7.8                                             500 μL             2M KCl                                                           1.25 mL             1M MgCl2                                                                                       500 μL Sample Buffer                                                                                   100mL             1M Tris-HCl pH 6.8                                     25 mL             2% SDS                                                          2 g             Glycerol                                                        10 mL             1M DTT                                                          2 mL             Bromophenol Blue                                       0.01 g SDS Running 5X Gel Buffer                                                                                 5 L                   2 L Tris                                                                         150 g               60 g SDS                                                                        25 g                 10 g Glycine (electrophoresis grade)                      720 g               288 g 20X SSC                                                                                     1 L                   2 L Sodium Chloride                                             175.32 g          350.64 g Sodium Citrate                                                88.23 g            176.46 g pH to 7.0                                                                                                                     5X SSC, .1% SDS:                                                                                     1 L             20X SSC                                                         250 mL             10% SDS                                                        10 mL 1X SSC, 1% SDS *The 1X SSC in the stock jug is a straight dilution, NOT this recipe!*                                                                                     1 L             20X SSC                                                         50 mL             10% SDS                                                        10 mL 1X STE                                                                                     1 L             NaCl                                                                5.84 g             1M Tris-HCl, pH 7.5                                     20 mL             0.5M EDTA                                                    20 mL STE + STET                                                                                     1 L             1M Tris-Cl, pH 8.0                                          10 mL             NaCl                                                                  5.84 g             0.5M EDTA                                                      2 mL             Triton X-100                                                   500 μl 50X TAE                                                                           1 L Tris                                                                  242 g Glacial Acetic Acid                                        57.1 mL 0.5M EDTA                                                    100 mL 10X TBE                                                                            1 L                   2 L       Tris                                                                  108.8 g            217.6 g                        Boric Acid                                                      55.0 g              110.0 g 0.5M EDTA                                                    40 mL              80 mL 5X TBS                                                                            1 L                   2 L Tris                                                                  24.2 g              48.4 g NaCl                                                                292.4 g            584.8 g  pH to 7.5 10X TE Buffer                                                                                     1 L 1M Tris-Cl, pH 7.5                                            100 mL 0.5M EDTA pH 8.0                                             20 mL Filter sterilize. TENG 1% Triton                                                                                     1 L             Gelatin  solution (25 g/500 mL)                 50 mL             NaCl                                                                8.77 g             0.5M EDTA                                                    10 mL             1M Tris, pH 7.3                                               10 mL             Triton X-100                                                   10 mL TENG 0.1% Triton                                                                                      1 L             Gelatin                                                           50 mL             NaCl                                                                8.77 g             0.5M EDTA                                                    10 mL             1M Tris, pH 7.3                                               10 mL             Triton X-100                                                   1 mL TENS                                                                                     1 L                   1M Tris, pH 7.4                                             10 mL             NaCl                                                                5.84 g             0.5M EDTA                                                    10 mL             SDS                                                                   5 g             β-mercapto Ethanol                                     10 mL             Filter sterilize 1X TNE + SDS                                                                                     100 mL             SDS                                                                 0.40 g             1M Tris, pH 8.0                                               1 mL             2 mL of NaCl (5 M)                                        5.84 g             0.5M EDTA                                                    200 ml 5X Transcription Buffer                                                                                     10 mL             1M Tris, pH 7.5                                               2 mL             0.2M MgCl2                                                                                1.5 mL             100mM Spermidone                                    1 mL             NaCl                                                                0.29 g 10X Transfer Buffer                                                                                     1 L             Tris                                                                  24.7 g                          Glycine                                                            112.6 g            Do not pH the transfer buffer 1X Transfer Buffer                                                                                         1 L             10X Transfer Buffer                                       100 mL             Methanol                                                         200 mL Add methanol to final 1X dilution only Tris-Saline                                                                                       1 L             1M Tris, pH 7.3                                               5 mL             NaCl                                                                  8.77 g Tris-Saline 0.1% Triton                                                                                     2 L             1M Tris, pH 7.2                                             5 mL             1M Tris, pH 7.4                                             5 mL             5M NaCl                                                         60 mL             Triton X-100                                                   2 mL 1X TTBS                                                                                     1 L             TBS                                                                 667 mL             Tween-20                                                      333 mL 4X Upper Tris                                                                                     1 L             1M Tris, pH 6.8                                            500 mL             SDS                                                                 4 g QIAGEN AND NUCLEIC ACID DETECTION BUFFERS Buffer 1                                                                                     100 mL             NaCl                                                                0.877 g             Tris                                                                  1.21 g             pH to 7.5 Buffer 2                                                                                     100 mL             NaCl                                                                2.34 g             Tris                                                                  1.21 g             pH to 7.5 Buffer A                                                                                     100 mL             Tris                                                                  1.576 g             NaCl                                                                1.754 g             pH to 9.5             Autoclave Buffer A, 0.3% Tween 20                                                                                     100 mL             Tris                                                                  1.576 g             NaCl                                                                1.754 g             Tween 20                                                        0.3 mL             pH to 9.5             Autoclave Qiagen Buffer P1                                                                                     107 mL             1M Tris, pH 8.0                                              5 mL                0.5M EDTA, pH 8.0                                       2 mL             H2O                                                                 100 mL             Autoclave Rnase A (add after autoclaving)                     0.0107 g Store at 4° C Qiagen Buffer P2                                                                                     100 mL             NaOH                                                             0.8 g             SDS                                                                 1.0 g Qiagen Buffer P3                                                                                     100 mL             Potassium Acetate                                        25.03 g             pH to 4.8 (use 12N)             Autoclave Qiagen Buffer QBT                                                                                     100 mL             NaCl                                                                4.38 g             Ethanol                                                            15 mL             MOPS                                                             1.05 g             Triton X-100                                                   150 ml             pH to 7.0 (use 1N) Qiagen Buffer QC                                                                                     100 mL             NaCl                                                                5.84 g             MOPS                                                             1.05 g             Ethanol                                                            15 mL             pH to 7.0, filter sterilize. Qiagen Buffer QF                                                                          100 mL NaCl                                                                  7.31 g MOPS                                                               1.03 g Ethanol                                                            15 mL PH to 8.2 Solution 1 (Plasmid prep)                                                                                     500 mL             1M glucose                                                    25 mL             1M Tris, pH 8.0                                              12.5 mL             0.5M EDTA, pH 8.0                                       10 mL Solution 2 (Plasmid prep)                                                                                     200 mL             NaOH                                                             1.6 g             SDS                                                                 2.0 g Solution 3 (Plasmid Prep)                                                                                     100 mL             Potassium Acetate                                        29.4 g             Acetic Acid, Galacial                                      11.5 mL MEDIA AND MEDIA SOLUTIONS C – Leu Media (Leucine dropout media)                                                                                     1 L             CSM-Leu-Ura                                                 0.67 g             Uracil                                                              0.024 g             Heat gently to dissolve.  Filter sterilize, store in cold room. Complete Medium + Interleukin-2 (IL-2)                                                                                       1 L             RPMI – 1640                                                   890 mL             FBS                                                                    100 mL             Penicillin/Streptomycin                                 10 mL Interleuken-2 (in 1 mL Sigma H20)                          200 μL PHA                                                                              1 vial 1M Glucose                                                                                     100 mL             Glucose                                                           18 g             Filter sterilize HAT medium (w/ puromycin)                                                                                     500 mL                                    100X mycophenolic acid (4 mg/mL)              5 mL             100X xanthine (25 mg/mL)                             5 mL             100X hypoxanthine (1.35 mg/mL)                 5 mL             FBS                                                                     50 mL             HEPES                                                                 5 mL             Antibiotics                                                         5 mL             Puromycin                                                        250 mg             DMEM                                                              425 mL LB Media                                                                                     1 L             NaCl                                                                10 g             Yeast extract                                                   5 g             Tryptone Peptone                                        10 g             Make 250 mL aliquots.  Autoclave. L-Glutamine                                                                                     505 mL             L-Glutamine (5.84 g/200 mL)                         5 mL             Media                                                              500 mL RPMI (10% FBS)                                                                                     550 mL             Na(CO3)2 (7.5%)                                           12.5 mL             10X RPMI                                                      55 mL             FBS                                                                 55 mL             1N NaOH                                                       4 mL             Pen/Strep                                                      5 mL SOC medium                                                                           250 mL Tryptone Peptone                                            5 g Yeast extract                                                   1.25 g NaCl                                                              0.15 g KCl                                                                0.05 g MgCl2                                                                                             0.51 g MgSO4                                                                                         2.5 mL 20% glucose (add after autoclaving)       10 mL YPD Media                                                                                        1 L             Yeast extract                                                   10 g             Peptone                                                           20 g             Dextrose                                                          20 g             (dextrorotatory form of glucose)             Filter sterilize POLYACRYLAMIDE AND AGAROSE GEL SOLUTIONS 30% Acrylamide/Bis-Acrylamide (37.5:1)                                                                                     500 mL             Acrylamide                                                     150 g             Bis-acrylamide                                                4 g 1% TAE Agarose Gel                                                                                     100 mL             1X TAE                                                          100 mL             Agarose or AquaPor LE                                 1 g             Microwave to melt.  Allow to cool slightly, pour, and let solidify. PAGE, Denaturing                                                                                                1 L                                                                         6%             8%           10%              12% 40% Acrylamide (19:1)                                150 mL      200          250               300 10X TBE                                                          100 mL      100          100               100 Urea                                                               410 g          410          410              410 PAGE, Non-Denaturing                                                                                     5.0%, 150 mL             8.0%, 150 mL             30% Acrylamide                                             25 mL                          40 mL              10X TBE                                                            15 mL                          15 mL                          Ammonium Persulfate                                    0.1 g                            0.1 g                10% SDS-PAGE, Denaturing, for Proteins                                                                                     Lower Layer               3% Stack             40% Acrylamide/Bis (29:1)                            2.5 mL                         500 ml             4X Lower Buffer                                              2.5 mL                         1.25 mL             dH2O                                                                 4.9 mL                         3.2 mL             Polymerize with:             Temed                                                              10 ml                            20 ml             10% APS                                                           50 ml                            20 ml             Makes 2 mini gels.                                              OTHER SOLUTIONS AND REAGENTS 10% (w/v) Ammonium Persulfate (APS)                                                                                     500 mL             Ammonium persulfate                                    50 g             Make 50 mL aliquots.  Store at -20°C. Cesium Chloride Solution                                                                                     100 mL             CsCl                                                                     96 g             50 mM Sodium Acetate, pH 5.0                     50 mL             DEPC                                                                  100 ml             Stir at 37°C overnight and autoclave. CsCl – EtBr Solution                                                                         18 mL CsCl                                                                1.0 g DNA solution                                               10 mL 10 mg/mL EtBr                                          8 mL Chloroform/ Isoamyl Alcohol Solution                                                                                     100 mL             Chloroform                                                     96 mL             Isoamyl Alcohol                                              4 mL Denaturing Solution                                                                                     1 L             NaCl                                                                87.66 g             NaOH                                                             20.0 g 0.1% DEPC-Treated H2O                                                                                  1 L dH2O                                                                   1000 mL Diethyl Pyrocarbonate (DEPC)                          1 mL Shake well for several minutes. Stir overnight with cap loosened at 37°C and autoclave. 10 mM dNTP Dilution *Use 100mM dNTP Set from Invitrogen*                                                                                     2.5 mL             GTP                                                                 250 ml             ATP                                                                 250 ml             TTP                                                                 250 ml             CTP                                                                 250 ml             dH2O                                                               1.5 mL             Combine dNTPs.  Divide into two tubes of 500 ml each.  Add 750 ml Sigma water to each tube.  Make 50 ml aliquots and store at -20°C. 500 mM EDTA, pH 8.0                                                                                     500 mL             EDTA Disodium Salt Dihydrate                    93.05 g             Heat to dissolve.  pH to 8.0             **EDTA does not dissolve until nearly pH 8.0 Gelatin                                                                                     500 mL             Gelatin                                                            25 g             Heat to dissolve.  Autoclave. 1% Gelatin/TBS                                                                                     100 mL             TBS                                                                 100 mL             Gelatin                                                            1.0 g             Microwave 1 minute on high 1% Gelatin/TTBS                                                                                     100 mL             TTBS                                                               100 mL             Gelatin                                                             1.0 g             Microwave 1 minute on high Guanidine Thiocyanate Solution                                                                                     1 L             Guanidine Thiocyanate                             508 g             Sarkosyl                                                          5 g             25 mM Sodium Citrate                               7.35 g             2-MercaptoEthanol                                     7 mL             Filter sterilize.  Cover bottle with aluminum foil. IPTG, 200 μg/mL                                                                          10 mL IPTG                                                               0.002 g Sigma H2O                                                      10 mL 10X Lithium Acetate                                                                           250 mL Lithium Acetate                                              25.5 g             pH to 7.5.  Filter sterilize. Lysozyme, 50 mg/mL                                                                                     10 mL             Lysozyme                                                        0.5 g             Store at -20°C. Neutralizing Solution                                                                                     1 L             NaCl                                                                87.66 g             Tris                                                                  121.0 g             pH to 7.5 Phenol/ Chloroform/ Isoamyl Alcohol Solution                                                                                     100 mL             Phenol (heat until liquid)                            50 mL                          Chloroform                                                     48 mL             Isoamyl alcohol                                               2 mL Pronase                                                                          100 mL Pronase                                                            2.0 g    1M Tris, pH 7.5                                               1 mL NaCl                                                                0.058 g             Self-digest for 1 hour at 37°C and store at -20°C. RTA 6/0 (0.3M salt)                                                                                       1 L             0.5M Na2HPO4                                               12.0 mL             0.5M NaH2PO4                                               88.0 mL             5M NaCl                                                         60.0 mL             Glycerol                                                          100.0 mL RTA 6/0 (1M salt, 25mM Imidazole)                                                                                     1 L             0.5M Na2HPO4                                               12.0 mL             0.5M NaH2PO4                                               88.0 mL             5M NaCl                                                         200.0 mL             Glycerol                                                          100.0 mL             Imidazole                                                        1.70 g             Wrap bottle in foil. RTA 6/0 (1M salt, 500mM Imidazole)                                                                                     1 L             0.5M Na2HPO4                                               12.0 mL             0.5M NaH2PO4                                               88.0 mL             5M NaCl                                                         100.0 mL             Glycerol                                                          100.0 mL             Imidazole                                                        34.03 g             Wrap bottle in foil. RTA  7/8                                                                            1 L 0.5M Na2HPO4                                               89.6 mL 0.5M NaH2PO4                                               10.4 mL 5M NaCl                                                         60.0 mL Glycerol                                                          100 mL RTA 7/8 (no salt or glycerol)                                                                            1 L 0.5M Na2HPO4                                               89.6 mL 0.5M NaH2PO4                                               10.4 mL           RTD                                                                                     1 L             1M Tris, pH 7.0                                           50 mL             Glycerol                                                        100 mL             5M NaCl                                                        5 mL             0.5M EDTA                                                    2 mL                            RT Mix                                                                                     20 mL             1M Tris, pH 7.8                                               1 mL             2M KCl                                                           750 μL             100 mM MgCl2                                                                        1 mL             NP-40                                                              100 μL             Poly (rA) ∙ p(dT) (1U/mL)                              1 mL             1M DTT                                                            40 μL             Sigma H20                                                       to volume             Make 1 mL aliquots.  Store at -20° C. 10% (w/v) SDS                                                                              500 mL SDS                                                                 50 g Sucrose Gradient                                                                                     100 mL             5M NaCl                                                         2 mL             1M Tris , pH 8.0                                              1 mL             0.5M EDTA, pH 8.0                                       200 ml             Sucrose                                                            w/v, varies according to need X-Gal (20 mg/mL)                                                                                     5 mL             X-Gal                                                              0.100 g             Dimethyl Foramide                                         5 mL                         (use glass pipette!)             Make ~500 μL aliquots.  Store in brown glass vials at -20° C