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        DNA Solutions (DNA分离)

        互联网

        4829

        Table of Contents

        10X TBE

        40% Acrylamide Stock

        Alkaline lysis solution

        10X TBE:

        216 g Tris base

        110 g boric acid

        16.6 g EDTA

        Add water to 2 liters.

        40% Acrylamide/Bisacrylamide (40% A&B):

        38 g Acrylamide (Kodak 5521)

        2 g N,N-Methylene-bisacrylamide (Kodak 8383)

        Dissolve in approx. 80 ml of double distilled water and then deionize by stirring with 5 g of Amberlite MB-1 (Sigma MB-1A) for 1 hour at room temperature. Suction filter to remove the Amberlite and adjust to a final volume of 100 ml with double distilled water. (store at 4deg.C).

        10x Agarose gel loading dye:

        15% Ficoll, 0.2% bromophenol blue, 0.2% xylene cyanol FF in double distilled water.

        1.5 g Ficoll (Sigma F-2637)

        0.02 g Bromophenol blue (Sigma B-0126)

        0.02 g xylene cyanole FF (Kodak T-1579)

        ddH2 O to 10 ml (store at -20deg.C).

        Alkaline lysis solution (NaOH/SDS) :

        0.2 N NaOH, 1% SDS in ddwater.

        20 ml of 1 N NaOH (or 0.8 gms)

        10 ml of 10% SDS (or 1.0 gms)

        ddH2 O to 100 ml (make fresh)

        15% Ammonium persulfate (APS):

        1.5 g APS (Kodak 11151)

        ddH2 O to 10 ml (store at 4deg.C).

        100 mM rATP (adenosine triphosphate) :

        619 mg dipotassium ATP (ICN 100004)

        sddH2 O to 10 ml (aliquot and store at -20℃).

        1 mg/ml BSA (bovine serum albumin) :

        5 mg BSA (Sigma A-9647)

        sddH2 O to 5 ml (aliquot and store at -20℃)

        100 mM calcium chloride (CaCl2) :

        1.48 g CaCl2-2H2 O

        ddH2 O to 100 ml

        autoclave to sterilize (store at 4deg.C).


        50 mM calcium chloride :

        0.74 g CaCl2-2H2 O

        ddH2 O to 100 ml

        autoclave to sterilize (store at 4deg.C).

        Deionized formamide :

        Stir formamide (Schwarz/Mann Biotech 800686) with Amberlite MB resin, 10 g. per 100 ml, for one hour to deionize; filter through Whatman 3MM paper, store in a dark bottle at room temperature or 4deg.C.

        10X denaturing buffer : 200 mM Tris-HCl, pH 9.5, 1 mM EDTA, 10 mM spermidine in double distilled water.

        2 ml 1 M Tris-HCl, pH 9.5

        20 μl 0.5 M EDTA, pH 8.0

        1 ml 100 mM spermidine

        ddH2 O to 10 ml (aliquot and store at -20℃)

        Diatomaceous earth (100 mg/ml): Suspend 10 g of diatomaceous earth (Sigma D-5384) in 100 ml of distilled water in a 100 ml graduated cylinder, and let it settle down for 3 hours. Decant the supernatant, and resuspend the pellet in 100 ml of 6 M guanidine HCl, pH 6.4, containing 50 mM Tris-HCl, 20 mM EDTA.

        Diatomaceous earth-wash buffer : 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 50% ethanol in double distilled water.

        10 ml 1 M Tris-HCl, pH 8.0

        2 ml 0.5 M EDTA, pH 8.0

        500 ml 100% ethanol (McCormick Distilling Co., Inc.)

        ddH2 O to 1 L

        1 M DTT (Dithiothreitol, Cleland's reagent):

        1.54 g DTT (Calbiochem 233155)

        ddH2 O to 10 ml (aliquot and store at -20℃).

        DNase-free RNase A

        20 mg/ml RNase A in 1 mM NaOAc, pH 4.5.

        200 mg RNase A (Sigma R-5500)

        3.3 μl 3 M NaOAc, pH 4.5

        ddH2 O to 10 ml

        boil for 10 minutes (aliquot and store at -20℃).

        0.5 M EDTA , pH 8.0 (disodium ethylenediamine tetraacetate):

        186.1 g Na2EDTA

        Dissolve in approx. 400 ml ddH2 O, adjust pH to 8.0 with 10 N NaOH, and adjust to 1 liter final volume with distilled water

        100 mM EDTA :

        20 ml 0.5 M EDTA

        80 ml ddH2 O

        100 ml

        95% ethanol/0.12 M NaOAc (ethanol/acetate):

        95 ml 100% ethanol

        4 ml 3 M NaOAc pH 4.5

        1 ml ddH2 O

        100 ml


        5 mg/ml ethidium bromide (EtBr):

        500 mg EtBr (Sigma E-8751)

        ddH2 O to 100 ml

        FE (formamide/EDTA): 5:1 (v/v) formamide:50 mM EDTA

        10 μl ddH2 O

        10 μl 100 mM EDTA

        100 μl deionized formamide

        make fresh

        10X Fill-in/Kinase buffer :

        (500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, 10 mM DTT, and 50 ug/ml BSA in double distilled water)

        5 ml 1 M Tris-HCl, pH 7.6

        1 ml 1 M MgCl2

        100 μl 1 M DTT

        500 μl 1 mg/ml BSA

        3.4 ml ddH2 O

        10 ml

        Fill-in Deoxynucleotide Preparation :

        To make 4 ml of the fill-in nucleotides at a concentration of 0.25 mM of each nucleotide, combine the following:

        500 μl PCR dNTPs (2 mM)

        3500 μl ddH2 O

        Aliquot this into 0.5 ml eppendorf tubes with 10 μl in each tube.

        To make 4 ml of these nucleotides at a final concentration of 0.25 mM from the stock 100 mM solutions, add the following:

        10 μl 100 mM dATP

        10 μl 100 mM dCTP

        10 μl 100 mM dGTP

        10 μl 100 mM dTTP

        3.6 ml ddH2 O

        Aliquot into 0.5 eppendorf tubes with 10 μl in each tube.

        To order these nucleotides, call Pharmacia at 1 800-526-3593. Order the dNTP set: 27-2035-01 dNTP set (100mM each dATP, dCTP, dGTP, dTTP- each in 250 μl volume) $174.00 for the set.

        20% glucose:

        20 g d-glucose

        ddH2 O to 100 ml

        filter sterilize

        6 M guanidine HCl , pH 6.4, containing 50 mM Tris-HCl, 20 mM EDTA:

        573.18 g guanidine-HCl (Sigma G-4505)

        50 ml 1 M Tris-HCl, pH 7.6

        40 ml 0.5 M EDTA, pH 8.0

        ddH2 O to 1 liter

        GET/lysozyme solution : (50 mM glucose, 25 mM Tris-HCl, pH 8.0, and 10 mM EDTA, pH 8.0 in double distilled water)

        0.9 g d-glucose

        2.5 ml 1 M Tris-HCl, pH 8.0

        2 ml 0.5 M EDTA, pH 8.0

        ddH2 O to 100 ml (filter sterilize and store at 4degC).

        Add 2 mg/ml lysozyme (Sigma L-6876) just before use.


        1 M HEPES, pH 7.5 :

        23.83 g HEPES (Sigma H-3375)

        ddH2 O to 100 ml

        adjust pH to 7.5 with potassium hydroxide (KOH) (store at 4deg.C).

        IPTG (isopropyl b-D-thiogalactopyranoside):25 mg/ml IPTG in double distilled

        water

        250 mg IPTG (Sigma I-5502)

        ddH2 O to 10 ml (aliquot and store at -20℃)

        1 M isocitrate (sodium salt-dihydrate) :

        29.41 g Na3isocitrate-2H2 O (Sigma C-7254)

        ddH2 O to 100 ml

        10x Kinase buffer : 500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 100 mM DTT in

        sterile double distilled water.

        5 ml 1 M Tris-HCl, pH 7.6

        1 ml 1 M MgCl2

        1 ml 1 M DTT

        sddH2 O to 10 ml (store in 25 ml aliquots at -20deg.C).

        Kanamycin sulfate (Kan): Stock of 5 mg/ml in sterile double distilled water (sddH2 O).

        0.5 g Kanamycin (Boehringer Mannheim 106 801)

        sddH2 O to 100 ml (Add to media for final conc. 20 ug/ml)

        1M KCl (potassium chloride):

        7.5 g KCl

        ddH2 O to 100 ml

        10x Ligation buffer : 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 10 mM DTT, 10 mM rATP, and 250 ug/ml bovine serum albumin (BSA) in sterile double distilled water.

        5 ml 1 M Tris-HCl, pH 7.6

        1 ml 1 M MgCl2

        1 ml 1 M DTT

        1 ml 100 mM rATP

        2.5 mg BSA

        sddH2 O to 10 ml (store in 25 ml aliquots at -20deg.C)

        Loading dye: 0.3% xylene cyanole FF, 0.3% bromophenol blue, 10 mM EDTA in deionized formamide.

        3 g xylene cyanole FF

        3 g bromophenol blue

        0.2 ml 0.5 M EDTA

        deionized formamide to 10 ml

        Lysozyme solution : 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 5 mg/ml lysozyme in sterile double distilled water.

        5 ml 1 M Tris-HCl, pH 8.0

        2 ml 0.5 M EDTA

        0.5 g lysozyme (Sigma L-6876)

        sddH2 O to 100 ml (make fresh)


        1 M MgCl2 (magnesium chloride):

        20.33 g MgCl2-6H2 O

        ddH2 O to 100 ml

        1 M MgSO4 (magnesium sulfate):

        12.04 g MgSO4

        ddH2 O to 100 ml (autoclave)

        1 M MnCl2 (manganese chloride):

        1.98 g MnCl2 (Sigma M-8530)

        ddH2 O to 10 ml (store protected from light)

        1 M MOPS:

        20.93 g MOPS (Sigma M-1254)

        Dissolve in 80 ml ddH2 O, adjust pH to 7.5 with 1 N NaOH, and bring volume to 100 ml.

        10X MOPS buffer : 400 mM MOPS, pH 7.5, 500 mM NaCl, 100 mM MgCl2 in double distilled water.

        400 μl 1 M MOPS, pH 7.5

        170 μl 3 M NaCl

        100 μl 1 M MgCl2

        330 μl ddH2 O

        1 ml

        2.7 M MOPS (acid form):

        5.65 g MOPS (acid form)

        ddH2 O to 10 ml

        MOPS-Acid buffer: 1.35 M MOPS (acid form), 100 mM MgCl2 in double distilled water.

        500 μl 2.7 M MOPS (acid form)

        100 μl 1 M MgCl2

        400 μl ddH2 O

        1 ml

        10X Mn2+/isocitrate buffer : 50 mM MnCl2, 150 mM isocitrate (Na salt), 25% glycerol in double distilled water

        50 μl 1 M MnCl2

        150 μl 1 M isocitrate

        250 μl glycerol

        550 μl ddH2 O

        1 ml


        10x MTBE (Modified Tris-borate-EDTA buffer): 1.3 M Tris, 0.4 M Boric acid, and 0.02 M EDTA in double distilled water.

        162 g Tris base

        27.5 g Boric acid

        9.3 g Na2EDTA

        ddH2 O to 1 L

        Nucleotide ordering information:

        100 mM dATP 27-2050-01 Pharmacia

        100 mM dCTP 27-2060-01 Pharmacia

        100 mM dGTP 27-2070-01 Pharmacia

        10 mM c7dGTP 988 537 Boehringer-Mannheim

        100 mM dTTP 27-2080-01 Pharmacia

        5 mM ddATP 27-2057-00 Pharmacia

        5 mM ddCTP 27-2065-00 Pharmacia

        5 mM ddGTP 27-2075-00 Pharmacia

        5 mM ddTTP 27-2085-00 Pharmacia

        20 mM dNTP stocks : Prepare from 100 mM stocks

        80 μl 100 mM dNTP

        40 μl 50:1 TE buffer

        280 μl ddH2 O

        400 μl

        5 mM dNTP stocks : Prepare from 20 mM stocks

        25 μl 20 mM dNTP

        10 μl 50:1 TE buffer

        65 μl ddH2 O

        100 μl

        2 mM dNTPs : 2 mM dATP, dCTP, dGTP, and dTTP in 5 mM Tris-HCl, pH 7.6, 0.1 mM EDTA

        100 μl 20 mM dATP

        100 μl 20 mM dCTP

        100 μl 20 mM dGTP

        100 μl 20 mM dTTP

        100 μl 50:1 TE buffer

        500 μl ddH2 O

        1 ml


        2 mM [alpha]-S-dNTPs : 2 mM [alpha]-S-dATP, [alpha]-S-dCTP, [alpha]-S-dGTP, and [alpha]-S-dTTP in 5 mM Tris-HCl, pH 7.6, 0.1 mM EDTA

        100 μl 20 mM [alpha]-S-dATP

        100 μl 20 mM [alpha]-S-dCTP

        100 μl 20 mM [alpha]-S-dGTP

        100 μl 20 mM [alpha]-S-dTTP

        100 μl 50:1 TE buffer

        500 μl ddH2 O

        1 ml

        3M NaCl (sodium chloride):

        17.53 g NaCl

        ddH2 O to 100 ml

        10N NaOH (sodium hydroxide):

        40 g NaOH

        ddH2 O to 100 ml.

        1N NaOH :

        10 ml 10 N NaOH

        ddH2 O to 100 ml

        9.5M NH4OAc (ammonium acetate):

        73.23 g NH4OAc

        ddH2 O to 100 ml

        8.0M NH4OAc :

        61.69 g NH4OAc

        ddH2 O to 100 ml

        10X PCR buffer : 500 mM KCl, 100 mM Tris-HCl, pH 8.5, 15 mM MgCl2 in sterile double distilled water

        5 ml 1 M KCl

        1 ml 1 M Tris-HCl, pH 8.5

        150 μl 1 M MgCl2

        ddH2 O to 10 ml

        PCR Deoxynucleotide Preparation : To make 12.5 ml of the PCR nucleotides at a concentration of 2 mM each nucleotide, combine the following:

        250 μl 100 mM dATP

        250 μl 100 mM dCTP

        250 μl 100 mM dGTP

        250 μl 100 mM dTTP

        11.5 ml ddH2 O

        Aliquot this into 25 tubes with 500 μl in each tube. This will keep the nucleotides from being frozed and thawed too much.

        To order these nucleotides, call Pharmacia at 1 800-526-3593. Order the dNTP set: 27-2035-01 dNTP set (100mM each dATP, dCTP, dGTP and dTTP- each in 250 μl volume)$174.00 for the set.


        20% PEG/2.5 M NaCl:

        7.3 g NaCl

        10 g PEG (MW=8000) (Fisher P156-3)

        Dissolve in 40 ml double distilled water by stirring, and then adjust the volume to 50 ml.

        50% PEG/0.5 M NaCl :

        5.85 g NaCl

        100 g PEG (MW=8000) (Fisher P156-3)

        Dissolve in 100 ml double distilled water by stirring, and then adjust the volume to 200 ml.

        PEG:TE rinse solution : 1:3 solution of 20% PEG containing 2.5M NaCl and 10 mM Tris-HCl, pH 8.0 containing 1 mM EDTA in double distilled water.

        250 μl 1 M Tris-HCl, pH 8.0

        50 μl 0.5 M EDTA

        12.5 ml 20% PEG/2.5 M NaCl.

        ddH2 O to 37.5 ml

        Phenol, TE-saturated: add an equal volume of 10 mM Tris-HCl, pH 7.5-8.0, 1 mM Na2EDTA to ultrapure phenol, mix well, allow phases to separate, remove and discard upper (aqueous) phase. Repeat until the pH of the aqueous phase is between 7.5-8.0 (store at 4℃).

        Phenol/chloroform/isoamyl alcohol (25:25:1):

        100 ml TE-saturated phenol

        100 ml chloroform

        4 ml isoamyl alcohol

        204 ml

        2M NaOAc (sodium acetate) :

        27.22 g NaOAc-3H2 O

        ddH2 O to 100 ml

        3M NaOAc , pH 4.5:

        408.24 g NaOAc-3H2 O

        Dissolve in approx. 800 ml ddH2 O , adjust pH to 4.8 with glacial acetic acid and bring to a final volume of 1 L with ddH2 O.

        10X Low Salt Restriction enzyme assay buffer : 100 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 10 mM DTT in sterile double distilled water.

        1 ml 1 M Tris-HCl, pH 7.6

        1 ml 1 M MgCl2

        0.1 ml 1 M DTT

        ddH2 O to 10 ml


        10X Medium Salt Restriction enzyme assay buffer : 500 mM NaCl, 100 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 10 mM DTT in sterile double distilled water.

        1.7 ml 3 M NaCl

        1 ml 1 M Tris-HCl, pH 7.6

        1 ml 1 M MgCl2

        0.1 ml 1 M DTT

        ddH2 O to 10 ml

        10X High Salt Restriction enzyme assay buffer : 1M NaCl, 500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 10 mM DTT in sterile double distilled water.

        3.3 ml 3 M NaCl

        5 ml 1 M Tris-HCl, pH 7.6

        1 ml 1 M MgCl2

        0.1 ml 1 M DTT

        ddH2 O to 10 ml

        10X SmaI Restriction enzyme assay buffer : 200 mM KCl, 100 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 10 mM DTT in sterile double distilled water.

        2 ml 1 M KCl

        1 ml 1 M Tris-HCl, pH 7.6

        1 ml 1 M MgCl2

        0.1 ml 1 M DTT

        ddH2 O to 10 ml

        RNase T1 : 100 U/ul in 50 mM Tris-HCl, pH 7.6

        100 μl RNase T1 (Sigma R-8251) (100,000 U/0.2 ml)

        25 μl 1 M Tris-HCl, pH 7.6

        375 μl ddH2 O

        500 μl

        10% SDS (sodium dodecyl sulfate):

        10 g SDS (Fisher S529-3)

        ddH2 O to 100 ml

        1X STB buffer : 25% sucrose and 50 mM Tris-HCl, pH 8.0 in double distilled water.

        25 g sucrose

        5 ml 1 M Tris-HCl, pH 8.0

        ddH2 O to 100 ml (filter sterilize and store at 4degC)

        Silanizing reagent : 5% solution of dichloro dimethyl silane in 1,1,1-trichloroethane.

        20X SSC (standard saline-citrate):

        17.53 g NaCl

        8.82 g sodium citrate

        Dissolve in approx. 80 ml ddH2 O, adjust pH to 7.0 with hydrochloric acid (HCl) and bring final volume to 100 ml.

        1X SSC (standard saline-citrate):

        5 ml 20X SSC

        95 ml ddH2 O

        100 ml

        5X Taq reaction buffer : 400 mM Tris-HCl, pH 9.0, 100 mM ammonium sulfate ((NH4)2SO4), pH 9.0, 25 mM MgCl2, and 5% dimethyl sulfoxide (DMSO) in sterile double distilled water.

        16 ml 1 M Tris-HCl, pH 9.0

        4 ml 1 M (NH4)2SO4, pH 9.0

        1 ml 1 M MgCl2

        2 ml DMSO

        17 ml ddH2 O

        40 ml

        5X Taq dilution buffer : 400 mM Tris-HCl, pH 9.0, 100 mM (NH4)2SO4, pH 9.0, and 25 mM MgCl2 in sterile double distilled water.

        16 ml 1 M Tris-HCl, pH 9.0

        4 ml 1 M (NH4)2SO4, pH 9.0

        1 ml 1 M MgCl2

        19 ml ddH2 O

        40 ml


        5X Taq "A" termination mix: 62.5 uM dATP, 250 uM dCTP, 375 uM c7dGTP, 250 uM dTTP and 1.5 mM ddATP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.

        20 μl 20 mM dATP

        80 μl 20 mM dCTP

        240 μl 10 mM 7deaza-dGTP

        80 μl 20 mM dTTP

        1920 μl 5 mM ddATP

        640 μl 50:1 TE buffer

        3420 μl sddH2 O

        6.4 ml

        5X Taq "C" termination mix : 250 uM dATP, 62.5 uM dCTP, 375 uM c7dGTP, 250 uM dTTP and 0.75 mM ddATP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.

        80 μl 20 mM dATP

        20 μl 20 mM dCTP

        240 μl 10 mM 7deaza-dGTP

        80 μl 20 mM dTTP

        960 μl 5 mM ddCTP

        640 μl 50:1 TE buffer

        4380 μl sddH2 O

        6.4 ml

        5X Taq "G" termination mix : 250 uM dATP, 250 uM dCTP, 94 uM c7dGTP, 250 uM dTTP and 0.125 mM ddGTP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.

        160 μl 20 mM dATP

        160 μl 20 mM dCTP

        120 μl 10 mM 7deaza-dGTP

        160 μl 20 mM dTTP

        320 μl 5 mM ddGTP

        1280 μl 50:1 TE buffer

        10600 μl sddH2 O

        12.8 ml

        5X Taq "T" termination mix: 250 uM dATP, 250 uM dCTP, 375 uM c7dGTP, 62.5 uM dTTP and 1.25 mM ddTTP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.

        160 μl 20 mM dATP

        160 μl 20 mM dCTP

        480 μl 10 mM 7deaza-dGTP

        40 μl 20 mM dTTP

        3200 μl 5 mM ddTTP

        1280 μl 50:1 TE buffer

        7480 μl sddH2 O

        12.8 ml

        20X TAE buffer : 0.8 M Tris, 0.4 M NaOAc, and 0.04 M Na2EDTA, and glacial acetic acid to pH 8.3 in double distilled water.

        96.9 g Tris base

        32.8 g NaOAc-3H2 O

        14.9 g Na2EDTA

        Dissolve in approx. 700 ml of double distilled water, adjust the pH to 8.3 with glacial acetic acid, and bring to 1 L with ddH2 O.

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