单组分溶液配制

Organic substances.

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".

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.

ATP

C₁₀H₁₄N₅O₁₃P₃Na₂ Mw=551.1 g/M; (store at -20^oC).

 0.1M ATP (pH 7.2): 5.71% (w/w) ATP, 84.86% (w/w) H₂O, 9.43% (w/w) 2M Tris base.

1. adjust to pH7.5 by 2M NaOH (~70-80µl);

2. prepare about 700µl, dilute 2000 times (it is the final dilution in the spectrofotometric cell), check the optical density:

   C[M]=A₂₅₉/15.4x10³=A_{259 1:2000}x0.130

3. adjust the final volume.

Betaine

monohydrate C₅H₁₁NO₂xH₂O; Mw=135.2g/M; (store at 4^oC).

p~1.073g/ml

Cresol red (Na)

50mM (store at +4, -20^oC):

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.

DTT

C₄H₁₀O₂S₂ Mw=154.2g/M; (store at -20^oC).

p~1.06g/ml

filter sterililize;

no DEPC treatment.

Preparation of dNTP's.

The quick protocol for ~100mM stock.

1. dilute all four dNTP's (250mg of each) in 3.676ml of H₂O;

2. + 424µl 5M NaOH;

3. check the pH: ~0.5µl on pH-paper.

Accurate protocol for 100mM stocks preparation.

1. 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

2. check the pH: ~0.5µl on pH-paper;

3. 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:

Concentration:

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

EDTA

C₁₀H₁₄O₈N₂Na₂x2H₂O; Mw=372.3g/M; pH = 8.0 (store at 4^oC).

p~1.096g/ml

EDTA is not soluble at acidic pH; it is necessary to add alkali gradually and to control pH;

do not treat by DEPC.

EtBr

Ethidium bromide, C₂₁H₂₀N₃Br; Mw=394.3g/M; (store at NT in the dark);

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.

Gelatin

(store at 4^oC).

sterilize by autoclaving.

Glucose

Guanidine HCl (GuHCl)

CH₅N₃xHCl, Mw=95.53g/M

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.

HEPES

p=1.064

HEPES, 1M, 1L

Imidazol

C₃H₄N₂, (store at 4^oC):

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).

PEG

H(OCH₂CH₂)_nOH; (store at 4^oC).

p=1.075.

PMSF

(store at -20^oC)

Retinoic acid

all trans-Retinoic acid, Tretinoin, light-sensitive, (store at -20^oC):

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

Sucrose

C₁₂H₂₂O₁₁, Mw=342.30g/M; 20^oC.

Tris Cl

C₄H₁₁O₃N; Mw=121.1g/M; (store at 4^oC).

1M: p=1.0406

 2M Tris base: 22.90%(w/w) Tris base, 77.10%(w/w) H₂O; p=1.058

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.

Temperature dependence of pH for Tris Cl 50mM.

TrisCl: 250ml 1M

Tricine

C₆H₁₃NO₅, Mw=179.2g/M;(store at 4^oC).

For 50ml:   

Triethanolamine

1M (store at 4^oC):

Urea

CH₄N₂O, Mw=60.06g/M;

solubility: at 25^oC: 10.49M, 5^oC: ~8M;

A₂₆₀(6M in H₂O)<0.06;

it is possible to take the "partial density of urea" as 0.763 in calculation of solutions.

Acids and alkalis.

NaOH

Mw=40g/M; (store at NT).

10M: 30.10%; p=1.329.

it is better to use plastic bottles for storage, because the alkali slightly solubilize the glass.

KOH

Mw=56.11g/M; (store at NT).

5M: 23.06%; p=1.217.

it is better to use plastic bottles for storage, because the alkali slightly solubilize the glass.

TCA

CCl₃CO₂H Mw=163.39g/M,(store at NT, in the dark, under the fume hood).

Detergents.

N-Lauroylsarcosine Na

(store at NT).

p=1.017.

SDS

Sodium dodecyl sulfate, sodium lauryl sulfate; [CH₃(CH₂)₁₀CH₂SO₄]Na; Mw=288.4g/M; (store at NT).

p = 1.018

weight under the fume hood (and better to wear the mask);

it is necessary to heat to 60-80^oC. to facilitate solubilization. Check pH. If it differs from neutral (~7.2-7.5) dramatically - adjust by diluted alkali / acid.

Organic solvents.

Phenol

C₆H₅OH; Mw=94.1g/M
p=1.054, t_m= 43, t_b= 182, pKa=10.0
solubility: 6.8¹⁶H₂O, unlimited ⁶⁶H₂O; unlimited EtOH

Preparation of "acidic" and "neutral" phenol.

a) from the distillation:

1. distill phenol under H₂O;

2. adjust water to the volume of about 1/10 of phenol phase;

3. add 8-hydroxyquinoline to 0.1% (relative to phenol phase) and bMeEtOH (2-mercaptoethanol) to 0.2% (relative to H₂O = 0.02% relative to phenol phase);

   ---- on this step you obtained the "acidic phenol". Store at -20^oC (~1 year); ----

4. add about the same volume of 0.2 M Tris-base to the phenol, mix ~0.5-1h;

5. throw away aqueous phase;

6. + 0.1V 0.1M Tris-Cl, pH 8.0

7. 0.2% bMeEtOH

8. mix ~0.5-1h;

9. store at 4^oC (in the dark) ~several months.

b) from the good commercial substance:

1. saturate phenol by the water (add about 1/5 V );

2. then, according to (a), from the p.2.

______________
* For RNA extraction it is better to saturate (just add) "acidic phenol" by the following buffer: (store at 4^oC):

check pH and adjust by diluted (1:100) acetic acid if necessary.

 there was note in the "Mitchell Group Methods" that acidic phenol is much more stable, then the neutral. They recommend preparing neutral phenol in small quantities only for a few weeks.

EtOH

CH₃CH₂OH; Mw=46.1g/M;

very hygroscopic, (store at NT in tightly closed bottle); t_пл.=-117.3^oC; t_кип.=78.5^oC. 
Conc.Stock300mlEtOH70%96%190gH2O mQ70ml

Preparation of 100% EtOH.

 test for the "100%" ethanol: add a drop of ethanol to the xylene. No mist should appear in the case of 100% ethanol.

1. dry CuSO₄ (at 65^oC, 1-2 days) to the practically white color;

2. mix about 1/4 of volume of the CuSO₄ with 3/4 volume of 96%EtOH;

3. mix intensively;

4. leave for 1-2 days (CuSO₄ will "take" water from ethanol).