Compound solutions.

About the recalculation of recipes for the arbitrary volume:

TE

1x; pH7.4; 7.6; 8.0; (store at 4^oC).

Conc. Stock 1ml 5ml 10ml 50ml 400ml Tris Cl 10mM 1M 10µl 50µl 100µl 500µl 4ml EDTA 1mM 0.5M 2µl 10µl 20µl 100µl 800µl H2O   mQ 988µl 4.94ml 9.88ml 49.4ml 395ml

10x:

Conc. Stock 1ml 5ml Tris Cl 0.1M 1M 0.1ml 0.5ml EDTA 10mM 0.5M 20µl 0.1ml H2O mQ   880µl 4.4ml

autoclave sterilization.

STE

1x; (store at 4^oC).

Conc. Stock 1ml 5ml 10ml 50ml NaCl 0.1M 5M 20µl 100µl 200µl 1.0ml Tris Cl, pH8.0 10mM 1M 10µl 50µl 100µl 0.5ml EDTA, pH8.0 1mM 0.5M 2µl 10µl 20µl 100µl H2O   mQ 0.968ml 4.84ml 9.68ml 48.4ml

10x:

Conc. Stock 1ml 5ml NaCl 1.0M 5M 0.2ml 1.0ml Tris Cl 0.1M 1M 0.1ml 0.5ml EDTA 10mM 0.5mM 20µl 0.1ml H2O mQ   680µl 3.4ml

autoclave sterilization.

SSC

20x; pH 7.0; (store at NT).

Conc. Stock 0.5L 1.5L 2L NaCl 3M 58.44g/M 87.66g 263.0g 350.6g Na3C6H5O7x 5.5H2O 0.3M 357.1g/M 53.57g 160.7g 214.3g H2O   mQ 440.3ml 1.32L 1.76L

p =1.103

TAE

1x pH=7.6:

Tris-acetate 40mM
EDTA 1mM

(store the large stock at 4^oC, working quantity (~100ml) at NT)

Conc. Stock 100ml 150ml 200ml 250ml Tris-base 2M 121.1g/M 24.22g 36.33g 48.44g 60.55g EDTA 0.05M 372.3g/M 1.862g 2.792g 3.723g 4.654g Acetic acid ~1M 17.4M ~5.71ml ~6.00g ~8.57ml ~9.00g ~11.42ml ~12.00g ~14.28ml ~15.00g H2O   mQ

p(50x)=1.0878

it is convenient to solubilize Tris and EDTA first, and only then add acid to adjust pH.

TBE

10x, pH ~8.3;(store at NT in clean bottle)

1x 10x Stock 1L Tris base 89mM 0.89M 121.14g/M 107.8g Boric acid 89mM 0.89M 61.83g/M 55.03g EDTA, pH 8.0 2mM 20mM 500mM 40ml 43.84g H2O     mQ 863.3ml

10x stock solution is stable at NT if to clean it through 0.4µm filter;

for PAAG it is used as 1x, for agarose gel - as 0.5x;

for agarose gels it is used for separation of small (< 200bp) fragments. Generally accepted, that the purification of fragments is more complex from TBE gels.

FGRB

50x (formaldehyde gel-running buffer), p=1.104g/ml (store at +4^oC):

Conc. Stock 0.5L 1.0L 2.0L MOPS (pH7.0) 1.0M 209.27g/M 104.64g 209.27g 418.54g AcONax3H2O 0.5M 136.08g/M 34.02g 68.04g 136.08g EDTAx2H2O 50mM 372.3g/M 9.31g 18.62g 37.23g H2O   mQ 404.0ml 808ml 1616ml

Conc. Stock 0.5L 1.0L 2.0L MOPS (pH7.0) 1.0M 209.27g/M 104.64g 209.27g 418.54g AcONa anhydrous 0.5M 82.04g/M 20.51g 41.02g 82.04g EDTA x 2H2O 50mM 372.3g/M 9.31g 18.62g 37.23g H2O   mQ 417.5ml 835ml 1670ml

adjust pH7.0 10N NaOH (per 500ml of buffer ~22ml), filter-sterilize through 0.22µm filter;

solution becomes yellow during the storage on the light, but it has no effect on the solution quality.

GTGB

20x /Glycerol tolerant gel buffer/ (store at +4^oC):

Conc. Stock 1L 1.5L 2.0L 2.5L Tris base 1.78M 121.14g/M 216g 324g 432g 540g Taurine 0.56M 125.15g/M 72g 108g 144g 180g Na2EDTAx2H2O 10.7mM 372.3 g/M 4g 6g 8g 10g H2O   mQ 802ml 1203ml 1604ml 2005ml

p=1.094

Taurine: C₂H₇NO₃S; Mw=125.15g/M.

TGB

Tris-Glycine buffer 5x, pH8.3, (store the main stock at +4^oC, working quantity at NT).

1x Conc. Stock 1L 1.5L Tris-base 25mM 125mM 121.1g/M 15.1g 22.71g Glycine 250mM 1.25M 75.05g/M 94g 141g SDS 0.1% 0.5% 10% 50ml 75ml H2O     mQ

Loading buffer

10x, (store at NT).

Conc. Stock 1ml 5ml 10ml 25ml Xilene cyanol 0.1% solid 1.0mg 5.0mg 10mg 25.0mg Bromph. blue 0.1% solid 1.0mg 5.0mg 10mg 25.0mg SDS 0.5% 10% 50µl 250µl 500µl 1.25ml EDTA, pH8.0 0.1M 0.5M 0.2ml 1.0ml 2.0ml 5.0ml Glycerol 50% 100% 0.5ml 0.625g 2.5ml 3.125g 5.0ml 6.25g 12.5ml 15.63g H2O   mQ 0.25ml 1.25ml 2.5ml 6.25ml

PEG-NaCl

(store at 4^oC).

Conc. Stock 50ml 100ml 150ml 200ml NaCl 1.6M 58.44g/M 4.675g 9.35g 14.03g 18.70g PEG-6000 13% solid 6.50g 13.00g 19.50g 26.00g

or:

Conc. Stock 50ml 100ml 150ml 200ml NaCl 1.6M 5M 16ml 18.97g 32ml 37.95g 48ml 56.92g 64ml 75.90g PEG-6000 13% 40% 16.25ml 32.50ml 48.75ml 65.00ml

SM

(store at 4^oC).

Conc. Stock 10ml 50ml 100ml 150ml NaCl 0.1M 5M 200µl 1.0ml 2.0ml 3.0ml MgSO4* 10mM 1M 100µl 0.5ml 1.0ml 1.5ml Tris Cl, pH7.5 50mM 1M 0.5ml 2.5ml 5.0ml 7.5ml Gelatin 0.01% 2% 50µl 0.25ml 0.5ml 0.75ml H2O   mQ 9.15ml 45.75ml 91.5ml 137.25ml

* - add after autoclaving;

autoclave sterilization.

STM

(store at 4^oC):

Conc. Stock 50ml NaCl 10mM 5M 100µl Tris Cl pH8.0 50mM 1M 2.5ml MgCl2 10mM 1M 500µl H2O   mQ 46.9ml

TM

(store at 4^oC):

Conc. Stock 50ml Tris Cl pH8.0 50mM 1M 2.5ml MgCl2 10mM 1M 500µl H2O   mQ 47.0ml

Denaturation buffer

(store at NT).

Conc. Stock 100ml 150ml 300ml 500ml 750ml NaCl 1.5M 58.44g/M 8.766g 13.15g 26.30g 43.83g 65.75g NaOH 0.5M 10M 5ml 6.65g 7.5ml 9.97g 15ml 19.94g 25ml 33.23g 37.5ml 49.84g     40g/M 2g 3g 6g 10g 15g

Neutralization buffer

(store at NT).

Conc. Stock 150ml 300ml 500ml 750ml Tris Cl, pH7.4 1M 121.1g/M 18.15g 36.3g 60.5g 90.75g NaCl 1.5M 58.44g/M 13.15g 26.30g 43.83g 65.75g HCl ~0.81M 11.6M/L ~10.5ml ~21ml ~35ml ~52.5ml EDTA (optional) 1mM 0.5M 0.3ml 0.6ml 1ml 1.5ml

Developer Rentgen 2

(store at NT in the dark).

500ml 800ml 1l Methol 1.1g 1.76g 2.2g Na2SO3 36.0g 57.6g 72.0g Hydroquinone 4.4g 7.04g 8.8g Na2CO3 24.0g 38.4g 48.0g KBr 2.0g 3.2g 4.0g H2O 490ml 784ml 980ml

solubilize in the presented order; r=1.115.

Fixer BKF 2

(store at NT).

500ml 800ml 1l Na2S2O3x 5H2O 130g 208g 260g NH4Cl 25g 40g 50g Sodium pyrosulfite 8.5g 13.6g 17g H2O 398ml 636.8ml 796ml

solubilize in the presented order; r=1.123.

Scintillation solution

(store at NT in the dark):

0.5L 1L PPO 2.50g 5.00g POPOP 0.05g 0.10g Toluene

I plasmid solution

(store at +4^oC)

Conc. Stock 50ml 100ml 150ml 200ml Glucose 50mM 2M 1.25ml 1.41g 2.5ml 2.825g 3.75ml 4.24g 5.0ml 5.56g Tris Cl, pH 8.0 25mM 1.0 M 1.25ml 2.5ml 3.75ml 5.0ml EDTA 10mM 0.5 M 1.0ml 2.0ml 3.0ml 4.0ml H2O   mQ 46.5ml 93ml 139.5ml 186ml

II plasmid solution

Conc. Stock 0.5ml 1ml 2.5ml 5.0ml 6.5ml 8.5ml 11ml NaOH 0.2N 1N 0.1ml 0.2ml 0.5ml 1.0ml 1.3ml 1.7ml 2.2ml SDS 1% 10% 0.05ml 0.1ml 0.25ml 0.5ml 0.65ml 0.85ml 1.1ml H2O   mQ 0.350ml 0.7ml 1.75ml 3.5ml 4.55ml 5. 95ml 7.7ml

Oligonucleotide gel staining mix

(store at NT).

Conc. Stock 100ml 200ml 300ml 500ml AcONa(5.2) 0.5M 3M 17ml 33ml 50.0ml 83ml Met. blue 0.04% 0.2% 20ml 40ml 60ml 100ml H2O   mQ 73ml 127ml 190ml 317ml

Mg²⁺2M

(store at NT).

Conc. Stock 200ml MgCl2x6H2O 1M 203.3g/M 40.66g MgSO4x7H2O 1M 246.48g/M 49.30g H2O   mQ 144.6g

p=1.173g/ml.

HSB (f⁺)

high SDS hybridization buffer (store the main stock at 4^oC, working - NT)

Conc. Stock 500ml SDS 7% solid 35g Formamid 50% 100% 250ml 283.3g SSC 5x 20x 125ml 145.3g Bloc. Reagent 2% solid 10g NaP (pH 7.0) 50mM 1M 25ml 27.6g N-Lauroylsarcosine Na 0.1% 10% 5ml 5.1g Salm.Sperm DNA 50µg/ml 10µg/µl 2.5ml H2O   mQ 49ml

p=1.111

Blocking Reagent - is the powder from "Roche Diagnostics GmBH; former Boehringer Mannheim" #1096176. Instead of 2% Blocking Reagent one may use 10x Denhardt or 2% non-fat dry milk.

it is possible to dissolve the Blocking Reagent only in high strength buffer (in this particular case in H₂O+SSC+NaP).

Salmon sperm DNA should be shredded and denaturated.

Restriction buffers 10x

À.

Conc. Stock 100µl 1ml Tris Ac (pH7.9 37oC) 33mM 1M 33µl 330µl MgAc 10mM 1M 10µl 100µl KAc 66mM 5M 13.2µl 132µl DTT 0.5mM 1M 0.5µl 5µl H2O   mQ 43.3µl 433µl

B.

Conc. Stock 100µl 1ml Tris Cl (pH 8.0 37oC)) 10mM 1M 10µl 100µl MgCl2 5mM 1M 5µl 50µl NaCl 100mM 5M 20µl 200µl b-MeEtOH 1mM 1M 1µl 10µl H2O   mQ 64µl 640µl

L.

Conc. Stock 100µl 1ml Tris Cl (pH 7.5 37oC) 10mM 1M 10µl 100µl MgCl2 10mM 1M 10µl 100µl DTT 1mM 1M 1µl 10µl H2O   mQ 79µl 790µl

M.

Conc. Stock 100µl 1ml Tris Cl (pH7.5 37oC) 10mM 1M 10µl 100µl NaCl 50mM 5M 10µl 100µl MgCl2 10mM 1M 10µl 100µl DTT 1mM 1M 1µl 10µl H2O   mQ 69µl 690µl

H.

Conc. Stock 100µl 1ml Tris Cl (pH7.5 37oC) 50mM 1M 50µl 50µl MgCl2 10mM 1M 10µl 100µl NaCl 100mM 5M 20µl 200µl DTT 1mM 1M 1µl 10µl H20   mQ 19µl 190µl

Denhardt solution 50x

(store at -20^oC)

Conc. Stock 50ml 300ml 1L Ficoll 400 1% solid 0.5g 3.0g 10g Polyvinilpyrolidone 1% solid 0.5g 3.0g 10g BSA (fr V) 1% solid 0.5g 3.0g 10g H2O   mQ 46.7ml 292.2ml 974ml

p=1.004

filter purification;

it is possible to prepare 100x.

PBS

pH(1x)=7.4, (store at NT or at 4^oC):

1x Conc. Stock 100ml 500ml KH2PO4 1.7mM 17mM 136.1g/M 0.231g 1.157g Na2HPO4 5.2mM 52mM 142g/M 0.738g 3.692g NaCl 150mM 1.5M 58.44g/M 8.77g 43.83g H2O   mQ

1x Conc. Stock 100ml 500ml KH2PO4 1.7mM 17mM 136.1g/M 0.231g 1.157g Na2HPO4x7H2O 5.2mM 52mM 268.1g/M 1.394g 6.971g NaCl 150mM 1.5M 58.44g/M 8.77g 43.83g H2O   mQ

autoclave sterilization;

pH is concentration dependent; thus check pH only for diluted 1x buffer;

pH of 10x should be ~6.8 (adjust by NaOH if necessary).

SSPE

pH 7.4

Conc. Stock 1L NaCl 3M 58.44g/M 175g NaH2PO4xH2O 200mM 137.99g/M 27.6g EDTA 20mM 372.24g/M 7.4g

adjust pH by 10N NaOH.

RNase A (DNase free)

(store at -20^oC, working quantities may be stored at 4^oC)

1. solubilize 10mg/ml in 1-50mM AcONa (pH4.5);
2. 15' in boiling water bath, then cool slowly to NT;

RNAse storage buffer

(store at -20^oC).

Conc. Stock 1ml 5ml 10ml Tris Cl, pH7.6 10mM 1M 10µl 50µl 100µl Glycerol 50% 100% 0.5ml 0.630g 2.5ml 3.152g 5.0ml 6.305g H2O   mQ 0.486g 2.43g 4.86g

Proteinase K

(store at +4^oC, long-term storage at –20^oC):
20mg/ml solution in 10mM Tris Cl, 1.0mM CaCl₂, 30% glycerol.

EDTA/glycogen

20x (store at -20^oC):

Conc. Stock 1.0ml EDTA 0.2M 0.5M 400 µl glycogen 1mg/ml 20mg/ml 50 µl H2O   mQ 550 µl

DNase RQ1 buffer

10x (store at -20^oC):

Conc. Stock 1ml Tris Cl (pH8.025oC) 0.4M 1M 400µl NaCl 0.1M 5M 20µl MgCl2 60mM 1M 60µl CaCl2 0.1M 1M 100µl H20   mQ 420µl

DNAse storage buffer

(store at -20^oC).

Conc. Stock 1ml 1.5ml 2.5ml 5ml 10ml Tris Cl, pH 7.0 20mM 1M 20µl 30µl 50µl 100µl 200µl NaCl 50mM 5M 10µl 15µl 25µl 50µl 100µl DTT 1mM 1M 1µl 1.5µl 2.5µl 5.0µl 10µl Glycerol 50% 100% 0.5ml 0.630g 0.75ml 0.9g 1.25ml 1.58g 2.5ml 3.15g 5.0ml 6.31g H2O   mQ 0.485g 0.73g 1.21g 2.42g 4.85g

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