单组分溶液配制
来自 : 蚂蚁淘
Organicsubstances.pKaandtemperaturedependenceofpHforcommonbuffers.ATP0.1MBetaine5MCresolred(Na)50mMDTT1M,2.2MdNTP’s100mMEDTA0.5MEtBr10mg/mlGelatin2%Glucose1;1.5;2MGuanidineHCl1-8MHEPES1MImidazol2MParaformaldehyde37%PEG40%PMSF100mMRetinoicacid10mMSucrose1;2;2.5MTrisCl1MTemperaturedependenceofpHforTrisCl.Tricine1MTriethanolamine1MUrea1-10M Acidsandalkalis.Summarytable.NaOH10M,1MKOH5MTCA100% Detergents.N-LauroylsarcosineNa10%SDS10% Organicsolvents.PhenolEthanolEtOHPreparationof100%EtOH.  Supplement. | Densitiesofsomesolutionsareavailableonthepage"Densitiesofacids,alkaliandorganicsubstances". Seealso:- Bufferdesign-onlinecalculationsoflargelistofbuffers(Prof.R.Beynon)onthepageJavabasedMolecularBIOLOGist"sWorkbenchEMBL.
- BufferCalculatoronthesiteofLabVelocity.Registrationisnecessary(free).
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Organicsubstances. pKaandtemperaturedependenceofpHforcommonbuffers. | Buffer | Mw | pKa20oC | Workingrange | deltapKaper10oC | | MES2-(N-morpholino)ethanesulfonicacid | 195.2 | 6.15 | 5.5-6.7 | -0.110 | | Bis-Trisbis(2-hydroxyethyl)iminotris(hydroxymethyl)methane | 209.2 | 6.5 | 5.8-7.2 | | | ADAN-(2-acetamido)-2-imidoaceticacid | 190.2 | 6.60 | 6.0-7.2 | -0.110 | | PIPESpiperasine-N,N"-bis(2-ethanesulfonicacid) | 302.4 | 6.80 | 6.1-7.5 | -0.085 | | ACES2-[(2-amino-2-oxoethyl)amino]ethanesulfonicacid | 182.2 | 6.90 | 6.1-7.5 | -0.200 | | MOPSO3-(N-morpholino)-2-hydroxypropanesulfonicacid | 225.3 | 6.9 | 6.2-7.6 | | | Imidazol-HCl | 68.08 | 6.95 | 6.2-7.8 | | | Bis-TrisPropane1,3-bis[tris(hydroxymethyl)methylamino]propane | 282.3 | 6.8* | 6.3-9.5 | | | BESN,N-bis(2-hydroxyethyl)-2-aminoethanesulfonicacid | 213.2 | 7.15 | 6.4-7.8 | -0.160 | | MOPS3-(N-morpholino)propanesulfonicacid | 209.3 | 7.20 | 6.5-7.9 | -0.013 | | TESN-tris(hydroxymethyl)methyl-2-aminoethanesulfonicacid | 229.2 | 7.50 | 6.8-8.2 | -0.200 | | HEPESN-(2-hydroxyethyl)piperazine-N"-(2-ethanesulfonicacid) | 238.3 | 7.55 | 6.8-8.2 | -0.140 | | DIPSO3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxyprpanesulfonicacid | 243.3 | 7.6 | 7.0-8.2 | | | TAPSO3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonicacid | 259.3 | 7.6 | 7.0-8.2 | | | HEPPSON-(2-hydroxyethyl)piperazine-N"-(2-hydroxypropanesulfonicacid) | 268.3 | 7.8 | 7.1-8.5 | | | POPSOpiperazine-N,N"-bis(2-hydroxypropanesulfonicacid) | 362.4 | 7.8 | 7.2-8.5 | | | TEAtriethanolamine | 149.2 | 7.8 | 7.3-8.3 | | | EPPSN-(2-hydroxyethyl)piperazine-N"-(3-propanesulfonicacid) | 252.3 | 8.0 | 7.3-8.7 | | | TricineN-tris(hydroxymethyl)methylglycine | 179.2 | 8.15 | 7.4-8.8 | -0.210 | | Tris(TRIZMA)tris(hydroxymethyl)aminomethane | 121.1 | 8.30 | 7.0-9.1 | -0.310 | | BicineN,N-bis(2-hydroxyethyl)glycine | 163.2 | 8.35 | 7.6-9.0 | -0.180 | | TAPSN-tris(hydroxymethyl)methyl-3-aminopropanesulfonicacid | 243.3 | 8.4 | 7.7-9.1 | | | Glycylglycine | | 8.40 | | -0.280 | | AMPSO3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid | 227.3 | 9.0 | 8.3-9.7 | | | CHES1-(N-cyclohexylamino)ethanesulfonicacid | 207.3 | 9.3 | 8.6-10.0 | | | CAPSO3-(cyclohexylamino)-2-hydroxy-1-propanesulfonicacid | 237.3 | 9.6 | 8.9-10.3 | | | AMP2-amino-2-methyl-1-propanol | 89.1 | 9.7 | 9.0-10.5 | | | CAPS3-(cyclohexylamino)-1-propanesulfonicacid | 221.3 | 10.4 | 9.7-11.1 | |
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*pKa=9.0forthesecondstageofdissociation.PreparationofdNTP"s. Thequickprotocolfor~100mMstock. - diluteallfourdNTP"s(250mgofeach)in3.676mlofH2O;
- +424µl5MNaOH;
- checkthepH:~0.5µlonpH-paper.
Accurateprotocolfor100mMstockspreparation. - addthenecessaryquantity(seetable)ofH2OandTrisbase1M(itispossIBLetotakethevolumeofsaltas~150µl);
| | Mw | V(H2O) | V(Trisbase1M) | V(final) | | dATP | C10H14N5O12P3Na2x3H2O | 589.2 | 3.24ml | 850µl | 4.24ml | | dGTP | C10H13N5O13P3Na3x2H2O | 609.2 | 3.55ml | 400µl | 4.10ml | | dCTP | C9H13N3O13P3Na3x2H2O | 569.1 | 3.44ml | 800µl | 4.39ml | | dTTP | C10H14N2O14P3Na3x2H2O | 584.1 | 3.73ml | 400µl | 4.28ml |
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- checkthepH:~0.5µlonpH-paper;
- checkthequalityandconcentration(itisusefultotakefinaldilution1:5000(~20µM).InthiscasetheopticaldensitywillbeintheregionofAm~0.3-themostaccuraterangeforspectrophotometer).Concentrationisc[mM]=k1:5000xAm.
Quality: | dATPpH7.0 | A250/A260=0.80+0.03A280/A260=0.12+0.02 | dCTPpH2.0(!) | A250/A260=0.45+0.03A280/A260=2.10+0.15A290/A260=1.60+0.10 | | dGTPpH7.5 | A250/A260=1.18+0.04A280/A260=0.67+0.03A290/A260=0.28+0.03 | dTTPpH7.5 | A250/A260=0.65+0.03A280/A260=0.73+0.03 |
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Concentration: | Mw | Am | pH | E | k(for1:5000) | | dATP[Na2] | 589.2 | 259 | 7.0 | 15.2x103 | 328.9 | | dGTP[Na3] | 609.2 | 253 | 7.5 | 13.7x103 | 365.0 | | dCTP[Na3] | 569.1 | 280 | 2.0(!!!) | 13.0x103 | 384.6 | | dTTP[Na3] | 584.1 | 267 | 7.5 | 9.6x103 | 520.8 | | | | | | | | ATP[Na4] | 595.1 | 259 | | 15.4x103 | | | CTP[Na4] | 571.1 | 280 | | 13.0x103 | | | GTP[Na4] | 611.1 | 252 | | 13.7x103 | | | UTP[Na4] | 572.1 | 262 | | 10.2x103 | |
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Concentration:c[M]=Amax/E;Amax=maximumofabsorption.
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Paraformaldehyde PFA(paraformaldehyde)37%(forhistochemistryitshouldbefreshlyprepared). 1.mixinthescrew-captube: PFA(solid)=0.37g,H2O=1.0mlNaOH(1N)=14µl; 2.solubilizeintheboilingwaterbath(toheat~1-3";untilpHwilldropto~7.0). TrisCl:250ml1M | VHCl | VHCl | pH | | 0 | 10.44 | | 1 | 9.54 | | 2 | 9.26 | | 3 | 9.08 | | 3.7 | 8.98 | | 5ml | | 8.76 | | 6ml | | 8.67 | | 10 | 8.4 | | 11ml | | 8.22 | | 14 | 8.13 | | 13.5ml | | 8.06 | | 14ml | | 8.04 | | 15ml | | 7.97 | | 17 | 7.92 | | 16.5ml | | 7.82 | | 16.7ml | | 7.8 | | 17ml | | 7.77 | | 18.3ml | | 7.63 | | 18.6ml | | 7.59 | | 20 | 7.75 | | 23 | 7.4 |
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Acidsandalkalis. | Name: | Formula: | Mw | %(w/w) | [M] | gin1Lofsubst. | p[g/ml] | ml/Lfor1Msol. | | Sodiumhydroxide | NaOH | 40 | 50% | 19.1 | 763 | 1.53 | 52.4 | | 30.1% | 10.0 | 400 | 1.329 | 100 | | 10% | 2.75 | 111 | 1.11 | 363.6 | Potassiumhydroxide | KOH | 56.1 | 50% | 13.5 | 757 | 1.52 | 74.1 | | 23.06% | 5.0 | 280.6 | 1.217 | 200 | | 10% | 1.94 | 109 | 1.09 | 515.5 | | Ammoniumhydroxide | NH4OH | 35.0 | 28% | 14.8 | 251 | 0.898 | 67.6 | | Aceticacid,glacial | CH3COOH | 60.05 | 99.5% | 17.4 | 1045 | 1.05 | 57.5 | | Aceticacid | 36% | 6.27 | 376 | 1.045 | 159.5 | | Formicacid | HCOOH | 46.02 | 90% | 23.4 | 1080 | 1.20 | 42.7 | | Hydrochloricacid | HCl | 36.5 | 36% | 11.6 | 424 | 1.18 | 86.2 | | 10% | 2.9 | 105 | 1.05 | 344.8 | | Nitricacid | HNO3 | 63.02 | 71% | 15.99 | 1008 | 1.42 | 62.5 | | 67% | 14.9 | 938 | 1.40 | 67.1 | | 61% | 13.3 | 837 | 1.37 | 75.2 | | Perchloricacid | HClO4 | 100.5 | 70% | 11.65 | 1172 | 1.67 | 85.8 | | 60% | 9.2 | 923 | 1.54 | 108.7 | | Phosphoricacid | H3PO4 | 98.0 | 85% | 18.1 | 1445 | 1.71 | 55.2 | | Sulfuricacid | H2SO4 | 98.1 | 96% | 18.0 | 1766 | 1.84 | 55.6 | Detergents. | Detergent | Tmelt | Mw[Da] | CMC | | monomer | micelle | %(w/v) | M | | Anionic | | SDS | 206 | 288 | 18,000 | 0.23 | 8.0x10-3 | | Cholate | 201 | 430 | 4,300 | 0.60 | 1.4x10-2 | | Deoxycholate | 175 | 432 | 4,200 | 0.21 | 5.0x10-3 | | Cationic | | C16TAB | 230 | 365 | 62,00 | 0.04 | 1.0x10-3 | | Amphoteric | | LysoPC | - | 495 | 92,000 | 0.0004 | 7.0x10-6 | | CHAPS | 157 | 615 | 6,150 | 0.49 | 1.4x10-3 | | Zwittergent3-14 | - | 365 | 30,000 | 0.011 | 3.0x10-4 | | Nonionic | | Octylglucoside | 105 | 292 | 8,000 | 0.73 | 2.3x10-2 | | Digitonin | 235 | 1,229 | 70,000 | - | - | | C12E8 | - | 542 | 65,000 | 0.005 | 8.7x10-5 | | LubrolPX | - | 582 | 64,000 | 0.006 | 1.0x10-4 | | TritonX-100 | - | 650 | 90,000 | 0.021 | 3.0x10-4 | | NonidentP-40 | - | 603 | 90,000 | 0.017 | 3.0x10-4 | | Tween80 | - | 1,310 | 76,000 | 0.002 | 1.2x10-5 |
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CMC-criticalmicelleconcentration.| Detergent | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 1-Stronglydenaturing;2-Dializable;3-Ionexchangeable,unsuitableforion-exchangechromatography;4-Complexesions;5-StrongA280;6-Assayinterference;7-Coldprecipitates;8-Highcost;9-Easeofpurification;10-RADIolabeled;11-Definitecomposition;12-Auto-oxidation | | Anionic | | SDS | + | + | + | + | - | - | + | - | + | + | + | - | | Cholate | - | + | + | + | - | - | - | - | + | + | + | - | | Deoxycholate | - | + | + | + | - | - | + | - | + | + | + | - | | Cationic | | C16TAB | + | + | + | - | - | - | + | - | + | - | + | - | | Amphoteric | | LysoPC | +/- | - | - | - | - | - | - | + | +/- | + | + | - | | CHAPS | - | + | - | - | - | - | - | + | + | - | + | - | | Zwittergent3-14 | +/- | +/- | - | - | - | - | - | + | + | - | + | - | | Nonionic | | Octylglucoside | - | + | - | - | - | - | - | + | - | + | + | - | | Digitonin | - | - | - | - | - | - | - | + | + | - | | | | C12E8 | - | - | - | +/- | - | - | - | + | - | + | - | + | | LubrolPX | - | - | - | +/- | - | +/- | - | - | - | + | - | + | | TritonX-100 | - | - | - | +/- | + | +/- | - | - | - | + | - | + | | NonidentP-40 | - | - | - | +/- | + | +/- | - | - | - | + | - | + | | Tween80 | - | - | - | +/- | - | +/- | - | - | - | + | - | + |
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C16TAB-hexadecyltrimethylammoniumbromide;LysoPC-lysophosphatidylcholine.*Sodiumcholateandsodiumdeoxycholateareunsolubleat<pH7.5orationicstrengthgreaterthen0.1%.SDSmayprecipitatebelow20oC;**Ionicdetergentsmayinduceproblemswithelectrophoresisandisoelectricfocusing;***Itispossibletoremovebydialysis;****Phenol-containingdetergents(forexampleTritonX-100andNP-40)precipitateduringFolinproteinassay(butdonotinterferewithBradfordproteinassay).Organicsolvents. Phenol C6H5OH;Mw=94.1g/Mp=1.054,tm=43,tb=182,pKa=10.0solubility:6.816H2O,unlimited66H2O;unlimitedEtOH Preparationof"acidic"and"neutral"phenol. a)fromthedistillation: - distillphenolunderH2O;
- adjustwatertothevolumeofabout1/10ofphenolphase;
- add8-hydroxyquinolineto0.1%(relativetophenolphase)andbMeEtOH(2-mercaptoethanol)to0.2%(relativetoH2O=0.02%relativetophenolphase);
----onthisstepyouobtainedthe"acidicphenol".Storeat-20oC(~1year);---- - addaboutthesamevolumeof0.2MTris-basetothephenol,mix~0.5-1h;
- throwawayaqueousphase;
- +0.1V0.1MTris-Cl,pH8.0
- 0.2%bMeEtOH
- mix~0.5-1h;
- storeat4oC(inthedark)~severalmonths.
b)fromthegoodcommercialsubstance: - saturatephenolbythewater(addabout1/5V);
- then,accordingto(a),fromthep.2.
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