单组分溶液配制

发布时间：2019-04-22 23:19 原文链接：单组分溶液配制

pK_a and temperature dependence of pH for common buffers.
ATP 0.1M
Betaine 5M
Cresol red (Na) 50mM
DTT 1M, 2.2M
dNTP’s 100mM
EDTA 0.5M
EtBr 10mg/ml
Gelatin 2%
Glucose 1; 1.5; 2M
Guanidine HCl 1-8M
HEPES 1M
Imidazol 2M
Paraformaldehyde 37%
PEG 40%
PMSF 100mM
Retinoic acid 10mM
Sucrose 1; 2; 2.5M
Tris Cl 1M
Temperature dependence of pH for TrisCl.
Tricine 1M
Triethanolamine 1M
Urea 1-10M

Acids and alkalis.

Summary table.
NaOH 10M, 1M
KOH 5M
TCA 100%

Detergents.

N-Lauroylsarcosine Na 10%
SDS 10%

Organic solvents.

Phenol
EthanolEtOH
Preparation of 100% EtOH.

Supplement.

Densities of some solutions are available on the page "Densities of acids, alkali and organic substances".

See also:

Buffer design - online calculations of large list of buffers (Prof. R.Beynon) on the page Java based Molecular Biologist's Workbench EMBL.
Buffer Calculator on the site of LabVelocity. Registration is necessary (free).

Organic substances.

pK_a and temperature dependence of pH for common buffers.

BufferMwpKa20oCWorkingrangedelta pKaper 10oCMES2-(N-morpholino)ethanesulfonic acid195.26.155.5-6.7-0.110Bis-Trisbis(2-hydroxyethyl)iminotris(hydroxymethyl)methane209.26.55.8-7.2 ADAN-(2-acetamido)-2-imidoacetic acid190.26.606.0-7.2-0.110PIPESpiperasine-N,N'-bis(2-ethanesulfonic acid)302.46.806.1-7.5-0.085ACES2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid182.26.906.1-7.5-0.200MOPSO3-(N-morpholino)-2-hydroxypropanesulfonic acid225.36.96.2-7.6 Imidazol - HCl68.086.956.2-7.8 Bis-Tris Propane1,3-bis[tris(hydroxymethyl)methylamino]propane282.36.8*6.3-9.5 BESN,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid213.27.156.4-7.8-0.160MOPS3-(N-morpholino)propanesulfonic acid209.37.206.5-7.9-0.013TESN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid229.27.506.8-8.2-0.200HEPESN-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)238.37.556.8-8.2-0.140DIPSO3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxyprpanesulfonic acid243.37.67.0-8.2 TAPSO3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid259.37.67.0-8.2 HEPPSON-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)268.37.87.1-8.5 POPSOpiperazine-N,N'-bis(2-hydroxypropanesulfonic acid)362.47.87.2-8.5 TEAtriethanolamine149.27.87.3-8.3 EPPSN-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)252.38.07.3-8.7 TricineN-tris(hydroxymethyl)methylglycine179.28.157.4-8.8-0.210Tris (TRIZMA)tris(hydroxymethyl)aminomethane121.18.307.0-9.1-0.310BicineN,N-bis(2-hydroxyethyl)glycine163.28.357.6-9.0-0.180TAPSN-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid243.38.47.7-9.1 Glycylglycine 8.40 -0.280AMPSO3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid227.39.08.3-9.7 CHES1-(N-cyclohexylamino)ethanesulfonic acid207.39.38.6-10.0 CAPSO3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid237.39.68.9-10.3 AMP2-amino-2-methyl-1-propanol89.19.79.0-10.5 CAPS3-(cyclohexylamino)-1-propanesulfonic acid221.310.49.7-11.1

* pKa=9.0 for the second stage of dissociation.

Cresol Red (Na salt) is a very convenient dye. At a concentration of about 0.2mM it is compatible with restriction digestion, PCR, sequencing. It may be used as marker for electrophoresis;
the color of the dye is pH-dependent (red, if pH>7.5; yellow, if pH <7.0). It is possible to use Cresol Red as pH-indicator (i) for denaturation of double-stranded DNA in sequencing, (ii) for silica-purification of DNA from agarose gel.

filter sterililize;
no DEPC treatment.

Preparation of dNTP's.

The quick protocol for ~100mM stock.

dilute all four dNTP's (250mg of each) in 3.676ml of H₂O;
+ 424µl 5M NaOH;
check the pH: ~0.5µl on pH-paper.

Accurate protocol for 100mM stocks preparation.

add the necessary quantity (see table) of H₂O and Tris base 1M (it is possible to take the volume of salt as ~150µl); MwV(H2O)V(Tris base 1M)V(final)dATPC10H14N5O12P3Na2x3H2O589.23.24ml850 µl4.24mldGTPC10H13N5O13P3Na3x2H2O609.23.55ml400 µl4.10mldCTPC9H13N3O13P3Na3x2H2O569.13.44ml800 µl4.39mldTTPC10H14N2O14P3Na3x2H2O584.13.73ml400 µl4.28ml
check the pH: ~0.5µl on pH-paper;
check the quality and concentration (it is useful to take final dilution 1:5000 (~20µM). In this case the optical density will be in the region of A_m~0.3 - the most accurate range for spectrophotometer).
Concentration is c[mM]=k_1:5000xA_m.

Quality:

dATPpH 7.0A250/A260=0.80+0.03A280/A260=0.12+0.02dCTPpH 2.0(!)A250/A260=0.45+0.03A280/A260=2.10+0.15A290/A260=1.60+0.10dGTPpH 7.5A250/A260=1.18+0.04A280/A260=0.67+0.03A290/A260=0.28+0.03dTTPpH 7.5A250/A260=0.65+0.03A280/A260=0.73+0.03

Concentration:

MwAmpHEk (for 1:5000)dATP[Na2]589.22597.015.2x103328.9dGTP[Na3]609.22537.513.7x103365.0dCTP[Na3]569.12802.0 (!!!)13.0x103384.6dTTP[Na3]584.12677.59.6x103520.8 ATP [Na4]595.1259 15.4x103 CTP [Na4]571.1280 13.0x103 GTP [Na4]611.1252 13.7x103 UTP [Na4]572.1262 10.2x103

Concentration: c[M]=A_max/E; A_max = maximum of absorption.

EDTA is not soluble at acidic pH; it is necessary to add alkali gradually and to control pH;
do not treat by DEPC.

soluble in H₂O, EtOH, chloroform;
concerning carcinogenic properties of EtBr. The only data that we found in the literature is that in mutagenic test (on bacteria) 90µg of EtBr gave the same results as the smoke concentrate from one cigarette.

sterilize by autoclaving.

solubility: at 25^oC - 8.54M, 5^oC - >8M;
A₂₆₀(6M in H₂O)<0.03;
it is possible to take the "partial density of GuHCl" as 0.763 in calculations of solutions.

Paraformaldehyde

PFA (paraformaldehyde) 37% (for histochemistry it should be freshly prepared).

1. mix in the screw-cap tube:

PFA (solid) = 0.37g,
H₂O = 1.0ml
NaOH (1N) = 14µl;

2. solubilize in the boiling water bath (to heat ~1-3'; until pH will drop to ~7.0).

stock solution is 10mM, working solution is freshly prepared 1mM in EtOH (it would be better to add pure EtOH to the control cells).

do not treat by DEPC;
sterilize by autoclaving;
pH of Tris-buffers is dependent from concentration. If to take 50mM solution as the original:
pH(500mM) => + 0.05
pH(5mM) => - 0.05
pH drops on 0.028 when the temperature rise on 1^oC.

单组分溶液配制

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