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STOCK SOLUTIONS AND MEDIA

STOCK SOLUTIONS AND MEDIA


Sterile water bottles
 200 ml, 500 ml, or 950 ml water. Autoclave. 

0.05 M CaCl2 (per 200 ml)
 CaCl2·2 H2O	1.5 g Add 198 ml water. 
Autoclave.

0.5 M CaCl2 (per 200 ml)
 CaCl2·2 H2O	15.0 g Add 188 ml water. Autoclave. 

0.1 M MgSO4 (per 200 ml)
 MgSO4 (anhydrous)	2.4 g Add 200 ml water. Autoclave. 

10% MSG (for 200 ml)
 Glutamic acid (Na)	20.0 g Add 188 ml water. Autoclave. 

40% Glucose (per 200 ml)
 Glucose(dextrose)	80.0 g Add water to 200 ml (~145-150 ml). Autoclave. 

10% CAA (per 200 ml)
 Casein Acid Hydrolysate	20.0 g Add 186 ml water. Autoclave. 

10% Yeast (per 200 ml)
 Yeast extract		20.0 g Add 150-160 ml water. Autoclave. Add 150 ul ß-mercaptoethanol after autoclaving. 

20X Tryptone/NaCl (per 200 ml)
 Tryptone		40.0 g NaCl			20.0 g Add 190 ml water. Autoclave. Add 150 ul ß-mercaptoethanol after autoclaving. 

10X M9 stock (for 200 ml)
 Na2HPO4 (anhydr.)	12.0 g KH2PO4			 6.0 g NaCl			 1.0 g NH4Cl			 2.0 g Add 196 ml H2O. Autoclave. Note: leave out NH4Cl to prepare 10X M9-N. 

10X M9 - N stock (for 200 ml)
 Na2HPO4 (anhydr.)	12.0 g KH2PO4			 6.0 g NaCl			 1.0 g Add 196 ml H2O. Autoclave. Note: leave out NH4Cl! 

100X P (for 200 ml)
 KH2PO4			18.1 g K2HPO4			 5.0 g pH to 6.3 with 10 N KOH! Add 192 ml H20. Autoclave. 

1X SM (for 200 ml)
 NaCl			1.16 g MgSO4			0.20 g Gelatin (2%)		1.00 ml or 20 mg Add 190 ml H2O, then add: Tris (1 M, pH 7.5)	10.0 ml Autoclave. 

2% Gelatin (per 500 ml)
 Gelatin			10 g Add 500 ml water. Autoclave. 

20% Succinate (for 500 ml)
 Succinic acid		100 g KOH			 90 g Add to 400 ml ice cold water. Measure pH to 6.4, filter sterilize. 

10X TBE (electrophoresis buffer)
 Tris base		108.0 g Na2EDTA			  9.3 g Boric Acid		 55.0 g Bring volume to 1 L with water. Use double distilled water. pH should be 8.3; if not adjust dropwise with conc. NaOH. 

Water agar stock (for 190 ml)
 Agar			3.0 g Add 190 ml H2O. Autoclave, store in 60C water bath. 

R agar stock
 			200 ml	500 ml  Tryptone........	  2.0 g	  5.0 g NaCl............	  1.0 g	  2.5 g Agar............	  3.0 g	  7.5 g Add H2O.........	194.0 ml 485.0 ml Autoclave, store in 60C water bath. 

LB agar 200 ml 1000 ml
 Tryptone ......		  2 g	 10 g NaCl .......... 	  1 g	  5 g Yeast extract .		  1 g	  5 g Agar ..........		  3 g	 15 g Add H2O ......		193 ml	990 ml Autoclave, store in 60C water bath. LB agar can also be made by adding 10 ml 10% yeast extract and 10 ml 20X Tryptone/NaCl to 190 ml WA. 

NZ 200 ml 500 ml
 NZ amine ....		2.0 g	5.0 g NaCl ........		1.0 g	2.5 g MgSO4 (anhydrous)..	0.2 g	0.5 g Add 10 M NaOH (1 drop/100 ml) after adding water. Autoclave. For agar media: Agar ........		2.4 g	6.0 g Stir before and after autoclaving. 

GYPC to WA stock: 200 ml 1000 ml
 Glucose (40%) .....	2.0 ml	10.0 ml Yeast extract (10%)	4.0 ml	20.0 ml 100X P ............	2.0 ml	10.0 ml CAA (10%) .........	4.0 ml	20.0 ml Succinate (20%)....	1.0 ml	 5.0 ml Add appropriate antibiotics. Pour plates. 

ORS minimal 200 ml 1000 ml
 10X M9 .........	 10 ml	 50 ml Succinate (20%).	  2 ml	 10 ml MSG (10%) ......	  2 ml	 10 ml Glucose (40%) ..	  1 ml	  5 ml MgSO4 (0.1 M) ..	  1 ml	  5 ml CaCl2 (0.5 M) ..	200 ul	  1 ml 1000X vitamins .	200 ul	1 ml Water ..........	184 ml	920 ml Omit succinate and add 10 ml glucose for storage medium. 

E. coli minimal 200 ml 1000 ml
 10X M9 ..........	20 ml	100 ml MgSO4 (0.1 M) ...	 2 ml	 10 ml CaCl2 (0.5 M) ...	 0.2 ml	  1 ml 1000X vitamins ..	 0.2 ml	  1 ml Glucose (40%) ...	 2 ml	 10 ml 

XC minimal 200 ml 1000 ml
 10X M9 ..........	5.0 ml	25 ml MgSO4 (0.1 M) ...	1.0 ml	 5 ml CaCl2 (0.5 M) ...	0.2 ml	 1 ml 200X 7 aa stock..	0.2 ml	 1 ml Glucose (40%) ...	0.5 ml	 2.5 ml Glycerol (50%) ..	4.0 ml	20 ml 

Nif plates
 10X Nif buffer		100 ml 10X trace elements	100 ml 1000X vitamins		  1 ml Sterile dd H2O		799 ml Agarose			  6 g Succinate (20%)		 20 ml (Succinate is already 0.4% from that in 10X nif buffer) 

10X Nif buffer 1000 ml
 K2HPO4			 34.02 g KH2PO4			 57.06 g Succinate (20%)		200.00 ml pH should be 6.4 after 10X dilution. 

1000X ORS vitamins
 Nicotinic acid		2.0 mg/ml Pantothenate		1.0 mg/ml Biotin			0.2 mg/ml Stir, filter sterilize. 

Trace elements
 MgSO4 (0.1 M)		 25 ml CaCl2 (0.5 M)		  5 ml Na2MoO4 (1 mg/ml)	  5 ml FeCl3 (1 mg/ml)		  5 ml Water			460 ml 

YEM (per 500 ml)
 K2HPO4			 0.5 g MgSO47 H2O		 0.2 g NaCl			 0.1 g Mannitol		10.0 g Yeast extract		 0.4 g Agar			15.0 g pH to 6.9 

YEM substitute (per 200 ml)
 To 180 ml WA stock: 10X M9			 8.0 ml Mannitol (20%)		20.0 ml Yeast extract (10%)	 1.6 ml 

SM10 selective media (for storage)
 10X versatile Mg	100 ml Thiamine		  5 ml Threonine		  5 ml Leucine			  5 ml Glucose (40%)		 10 ml Water			875 ml 

Amino acids
 SM10 stocks:		Thiamine (0.337%) 			Threonine (0.71%) 			Leucine   (0.79%) Klebsiella:		Histidine (0.31%) 

10X versatile Mg (for 200 ml)
 MgSO4			2.0 ml Thiamine (1 mg/ml)	2.0 ml CaCl2			0.2 ml 

200X PLT (for 200 ml)
 Proline			9.20 g Leucine			1.60 g Thiamine		0.67 g Autoclave. 

Mg PLT
 10X versatile Mg	100 ml 200X PLT		  5 ml 40% glucose		 10 ml Water			885 ml  

Z buffer (lacZ assays)
 			200 ml	1000 ml Na2HPO47 H2O ....	3.22 g	16.10 g NaH2PO4H2O ......	1.10 g	 5.50 g KCl ..............	0.15 g	 0.75 g MgSO4 (0.1 M) ....	1.00 ml	 0.20 ml ß-mercaptoethanol.	2.70 ml	 0.54 ml Filter sterilize. 

ONPG stock
 4 mg/ml in phosphate buffer (pH 7) 

Phosphate buffer (pH 7.0) per 100 ml:
 Na2HPO47 H2O 		1.64 g NaH2PO4H2O 		1.38 g 

0.2 M Na2CO3 (100 ml)
 Na2CO3 (anhydr.)	10.6 g 

Jensen's Medium (Sesbania)
 10X Macro nutrients (1000 ml):  K2HPO43 H2O		 2.6 g  NaCl			 2.0 g  MgSO4			 0.97 g  CaCl2			10.0 g  FeCl36 H2O		 1.4 g  KNO3			 3.0 g 

Jensen's Medium (Sesbania)
 1X solution (1000 ml):  K2HPO43 H2O		0.20 g  NaCl			0.20 g  MgSO47 H2O		0.20 g  CaHPO4			1.00 g  FeCl36 H2O		0.10 g  or Fe EDTA		0.26 g  KNO3 .(only for 10X)	3.00 g 

Jensen's Medium (1942)
 see Vincent 1970 p. 75. per Liter:  K2HPO43 H2O		0.2 g  NaCl			0.2 g  MgSO47 H2O		0.2 g  CaHPO4			1.0 g  FeCl36 H2O		0.1 g   or Fe-EDTA		0.26 g Dilute 1:5 when used. 15 ml diluted medium/pouch. 

1000X Micronutrients (200 ml)
 H3BO3   		572 mg MnCl2			406 mg ZnSO4H2O		 44 mg CuSO45 H2O		 16 mg NaMO4BH2O		18 mg Co2+			20 mg Ni2+			20 mg 

Nodule grinding buffer (500 ml)
50 mM KH2PO4 (pH 7.6), 0.15 M NaCl
 K2HPO4 (1M)	 3.25 ml KH2PO4 (1M)	21.75 ml NaCl		 4.38 gm Add to 450 ml distilled water, pH to 7.6, and adjust volume to 500 ml. 

Rapidyne germicide
 For working solution: Dilute concentrated stock at 0.6ml/L. 

1 M Tris-Cl (500 ml)
 To 400 ml, add: Tris base	60.5 g Add conc. HCl to pH bring to volume. 

Tris buffer mixtures for pH
 pH	Tris HCl	Tris base 7.0	.923		.077 7.1	.905		.095 7.2	.90		.10 7.3	.865		.135 7.4	.84		.16 7.5	.82		.18 7.6	.78		.22 7.7	.74		.26 7.8	.67		.33 7.9	.63		.37 8.0	.50		.50 

Southern Transfer solutions
 I. 0.25 N HCl: 	21.5 ml conc. HCl (11.6N) to 1 liter volume. II. 0.5 M NaOH, 1.5 M NaCl: 	87.7 g NaCl, 20.0 g NaOH to 1 liter volume. III. 1.0 M Tris, 1.5 M NaCl: 	121.1 g Tris base, 87.7 g NaCl, pH to 8.0 with conc. HCl (~50 	ml), adjust to 1 liter volume. IV. 20X SSC: 	175.3 g NaCl, 88.2 g Na3citrate, pH to 7.0 with conc. HCl (~1 	drop), adjust to 1 liter volume. 

Random-primer oligonucleotide labelling
 Mix: 	9.00 ul DNA (not more than 200 ng) 	1.25 ul hexamers Boil, then place on ice, then add: 	5.0  ul HEPES buffer 	5.0  ul nucleotides [in 250 mM Tris (pH 8), 25 mM MgCl2, 50 mM 	ß-mercaptoethanol] 	1.0 ul BSA (40 mg/ml) 	0.5 ul Klenow 	5.0 ul -32P CTP Let reaction go at room temperature for at least 4 hours, or overnight.  

MS MEDIUM FOR ARABIDOPSIS

To 990 ml H2O add: Sucrose ........... 10.0 g MOPS .............. 0.5 g Agar .............. 8.0 g Adjust pH to 5.7 with 1 M KOH. Autoclave. When cooled add sterile stock solutions of: KNO3............... 20.0 ml 940 mM (95.00 g/L) NH4NO3............. 20.0 ml 1000 mM (80.04 g/L) KH2PO4............. 12.5 ml 100 mM (13.60 g/L) MgSO47H2O......... 15.0 ml 100 mM (24.65 g/L) CaCl27H2O......... 10.0 ml 300 mM (44.00 g/L) Glycine............ 2.67 ml 10 mM Nicotinate......... 0.4 ml 10 mM Thiamin............ 0.02 ml 10 mM PyridoxineHCl..... 0.24 ml 10 mM Myoinositol........ 5.5 ml 100 mM FeSO47H2O ........ 1.0 ml (2.780 g/100 ml) Na2EDTA ........... 1.0 ml 100 mM (3.720 g/100 ml) MnSO44H2O......... 1.0 ml 100 mM (1.690 g/100 ml) ZnSO44H2O......... 3.0 ml 10 mM (0.288 g/100 ml) H3BO3 ............. 1.0 ml 100 mM (0.618 g/100 ml) KI................. 0.5 ml 10 mM (0.166 g/100 ml) CuSO45H2O ........ 0.01 ml 10 mM (0.170 g/100 ml) Na2MoO42H2O ...... 0.1 ml 10 mM (0.242 g/100 ml) CoCl26H2O ........ 0.01 ml 10 mM (0.238 g/100 ml)

B5 MEDIUM FOR ARABIDOPSIS

To 990 ml H2O add: Sucrose ........... 20.0 g MOPS .............. 0.5 g Agar .............. 8.0 g Adjust pH to 5.7 with 1 M KOH. Autoclave. When cooled add sterile stock solutions of: KNO3............... 26.3 ml 940 mM (95.00 g/L) NaH2PO4H2O........ 11.0 ml 100 mM MgSO47H2O......... 10.0 ml 100 mM (24.65 g/L) (NH4)2SO3.......... 10.0 ml 100 mM CaCl27H2O......... 3.3 ml 300 mM (44.00 g/L) Myoinositol........ 5.5 ml 100 mM Thiamine........... 3.0 ml 10 mM Nicotinate......... 0.8 ml 10 mM PyridoxineHCl..... 0.48 ml 10 mM 10X Micronutrients. 10.0 ml (see below) Sequestrene 330 Fe. 10.0 ml (0.28 g/100 ml) For Callus Inducing Medium (CIM) add: 2,4-D..................... 0.50 ml Kinetin................... 0.05 ml For Shoot Inducing Medium (SIM) add: IAA....................... 0.15 ml 6-dimethylaminopurine..... 5.00 ml

10X Micronutrients (per 100 ml)
 MnSO44H2O.........	 0.6  ml  100 mM (1.690 g/100 ml) ZnSO44H2O.........	10.07 ml   10 mM (0.288 g/100 ml) H3BO3 .............	 4.9  ml  100 mM (0.618 g/100 ml) KI.................	 4.5  ml   10 mM (0.166 g/100 ml) CuSO45H2O ........	 2.3  ml   10 mM (0.170 g/100 ml) Na2MoO42H2O ......	 1.0  ml   10 mM (0.242 g/100 ml) CoCl26H2O ........	 1.0  ml   10 mM (0.238 g/100 ml) 

LB (Luria-Bertani) Broth
 Bacto-Tryptone		10 g Bacto-Yeast Extract	 5 g NaCl			 5 g H2O up to 1L  If desired, adjust the pH 7.5 with NaOH. 

CA (Complete Agar)
  Casein Hydrolysate	10 g Yeast Extract		 5 g K2HPO4	 4 g Agar			15 g H2O up to 1 L 

Stab Agar
  Nutrient Broth		10 g NaCl			 5 g Agar			 6 g H2O up to 1 L  Mix well and autoclave for 15 min at 121 C. 

S.O.C. (from BRL)
 			1X 100 ml 2 % Bacto-Tryptone	2.0 g  0.5 % Yeast Extract	0.5 g  10 mM NaCl		2.92 g 2.5  mM KCl		3.73 g 20 mM MgCl2		20.2 g 20 mM MgSO4		24.6 g 20 mM Glucose		9.0 g  Mix above, bring to 100 ml, and filter through a sterile 0.45 µm filter.  Store the stock solution at -20 C or -70 C. 

Bacillus Thuringiensis Medium
 Component		G/L  Glucose			3.0 (NH4)2SO4		2.0 Yeast Extract		2.0 K2HPO4 3H2O		0.5 MgSO4*7H2O		0.2 CaCl2*2H2O		0.08 MnSO4*4H2O		0.05  Add componenets to distilled water. Mix throughly and bring pH to 7.3. Autoclave for 15 min at 15 psi -- 121C