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Organicsubstances.

pK_aandtemperaturedependenceofpHforcommonbuffers.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).

Organicsubstances.

pK_aandtemperaturedependenceofpHforcommonbuffers.

Buffer	Mw	pK_a20^oC	Workingrange	deltapK_aper10^oC
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

*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

checkthepH:~0.5µlonpH-paper;
checkthequalityandconcentration(itisusefultotakefinaldilution1:5000(~20µM).InthiscasetheopticaldensitywillbeintheregionofA_m~0.3-themostaccuraterangeforspectrophotometer).Concentrationisc[mM]=k_1:5000xA_m.

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

Concentration:

	Mw	A_m	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

Concentration:c[M]=A_max/E;A_max=maximumofabsorption.

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

Acidsandalkalis.

Detergents.

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	CH₃COOH	60.05	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
Hydrochloricacid	HCl	36.5	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
Perchloricacid	HClO₄	100.5	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

Detergent	T_melt	Mw[Da]		CMC
Detergent	T_melt	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

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	-	-	-	+/-	-	+/-	-	-	-	+	-	+

C₁₆TAB-hexadecyltrimethylammoniumbromide;LysoPC-lysophosphatidylcholine.*Sodiumcholateandsodiumdeoxycholateareunsolubleat<pH7.5orationicstrengthgreaterthen0.1%.SDSmayprecipitatebelow20^oC;**Ionicdetergentsmayinduceproblemswithelectrophoresisandisoelectricfocusing;***Itispossibletoremovebydialysis;****Phenol-containingdetergents(forexampleTritonX-100andNP-40)precipitateduringFolinproteinassay(butdonotinterferewithBradfordproteinassay).

Organicsolvents.

Phenol

C₆H₅OH;Mw=94.1g/Mp=1.054,t_m=43,t_b=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-20^oC(~1year);----
addaboutthesamevolumeof0.2MTris-basetothephenol,mix~0.5-1h;
throwawayaqueousphase;
+0.1V0.1MTris-Cl,pH8.0
0.2%bMeEtOH
mix~0.5-1h;
storeat4^oC(inthedark)~severalmonths.

b)fromthegoodcommercialsubstance:

saturatephenolbythewater(addabout1/5V);
then,accordingto(a),fromthep.2.

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