Archived Protocol

<BODY BACKGROUND="../gif/chalk.gif" TEXT=00009C LINK=8E236B VLINK=CC3299 ALINK=FF1CAE>  <TITLE>STOCK SOLUTIONS AND MEDIA</TITLE> <HEAD><DL><DD> <H2>STOCK SOLUTIONS AND MEDIA</H2> </HEAD> <HR> Sterile water bottles <PRE> 200 ml, 500 ml, or 950 ml water. Autoclave. </PRE><HR>0.05 M CaCl2 (per 200 ml) <PRE> CaCl2·2 H2O 1.5 g Add 198 ml water. Autoclave. </PRE><HR>0.5 M CaCl2 (per 200 ml) <PRE> CaCl2·2 H2O 15.0 g Add 188 ml water. Autoclave. </PRE><HR>0.1 M MgSO4 (per 200 ml) <PRE> MgSO4 (anhydrous) 2.4 g Add 200 ml water. Autoclave. </PRE><HR>10% MSG (for 200 ml) <PRE> Glutamic acid (Na) 20.0 g Add 188 ml water. Autoclave. </PRE><HR>40% Glucose (per 200 ml) <PRE> Glucose(dextrose) 80.0 g Add water to 200 ml (~145-150 ml). Autoclave. </PRE><HR>10% CAA (per 200 ml) <PRE> Casein Acid Hydrolysate 20.0 g Add 186 ml water. Autoclave. </PRE><HR>10% Yeast (per 200 ml) <PRE> Yeast extract 20.0 g Add 150-160 ml water. Autoclave. Add 150 ul ß-mercaptoethanol after autoclaving. </PRE><HR>20X Tryptone/NaCl (per 200 ml) <PRE> Tryptone 40.0 g NaCl 20.0 g Add 190 ml water. Autoclave. Add 150 ul ß-mercaptoethanol after autoclaving. </PRE><HR>10X M9 stock (for 200 ml) <PRE> 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. </PRE><HR>10X M9 - N stock (for 200 ml) <PRE> Na2HPO4 (anhydr.) 12.0 g KH2PO4 6.0 g NaCl 1.0 g Add 196 ml H2O. Autoclave. Note: leave out NH4Cl! </PRE><HR>100X P (for 200 ml) <PRE> KH2PO4 18.1 g K2HPO4 5.0 g pH to 6.3 with 10 N KOH! Add 192 ml H20. Autoclave. </PRE><HR>1X SM (for 200 ml) <PRE> 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. </PRE><HR>2% Gelatin (per 500 ml) <PRE> Gelatin 10 g Add 500 ml water. Autoclave. </PRE><HR>20% Succinate (for 500 ml) <PRE> Succinic acid 100 g KOH 90 g Add to 400 ml ice cold water. Measure pH to 6.4, filter sterilize. </PRE><HR>10X TBE (electrophoresis buffer) <PRE> 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. </PRE><HR>Water agar stock (for 190 ml) <PRE> Agar 3.0 g Add 190 ml H2O. Autoclave, store in 60C water bath. </PRE><HR>R agar stock<PRE> 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. </PRE><HR>LB agar 200 ml 1000 ml <PRE> 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. </PRE><HR>NZ 200 ml 500 ml <PRE> 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. </PRE><HR>GYPC to WA stock: 200 ml 1000 ml <PRE> 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. </PRE><HR>ORS minimal 200 ml 1000 ml <PRE> 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. </PRE><HR>E. coli minimal 200 ml 1000 ml <PRE> 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 </PRE><HR>XC minimal 200 ml 1000 ml <PRE> 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 </PRE><HR>Nif plates <PRE> 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) </PRE><HR>10X Nif buffer 1000 ml <PRE> K2HPO4 34.02 g KH2PO4 57.06 g Succinate (20%) 200.00 ml pH should be 6.4 after 10X dilution. </PRE><HR>1000X ORS vitamins <PRE> Nicotinic acid 2.0 mg/ml Pantothenate 1.0 mg/ml Biotin 0.2 mg/ml Stir, filter sterilize. </PRE><HR>Trace elements <PRE> 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 </PRE><HR>YEM (per 500 ml) <PRE> 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 </PRE><HR>YEM substitute (per 200 ml) <PRE> To 180 ml WA stock: 10X M9 8.0 ml Mannitol (20%) 20.0 ml Yeast extract (10%) 1.6 ml </PRE><HR>SM10 selective media (for storage) <PRE> 10X versatile Mg 100 ml Thiamine 5 ml Threonine 5 ml Leucine 5 ml Glucose (40%) 10 ml Water 875 ml </PRE><HR>Amino acids <PRE> SM10 stocks: Thiamine (0.337%) Threonine (0.71%) Leucine (0.79%) Klebsiella: Histidine (0.31%) </PRE><HR>10X versatile Mg (for 200 ml) <PRE> MgSO4 2.0 ml Thiamine (1 mg/ml) 2.0 ml CaCl2 0.2 ml </PRE><HR>200X PLT (for 200 ml) <PRE> Proline 9.20 g Leucine 1.60 g Thiamine 0.67 g Autoclave. </PRE><HR>Mg PLT <PRE> 10X versatile Mg 100 ml 200X PLT 5 ml 40% glucose 10 ml Water 885 ml </PRE><HR>Z buffer (lacZ assays) <PRE> 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. </PRE><HR>ONPG stock <PRE> 4 mg/ml in phosphate buffer (pH 7) </PRE><HR>Phosphate buffer (pH 7.0) per 100 ml: <PRE> Na2HPO47 H2O 1.64 g NaH2PO4H2O 1.38 g </PRE><HR>0.2 M Na2CO3 (100 ml) <PRE> Na2CO3 (anhydr.) 10.6 g </PRE><HR>Jensen's Medium (Sesbania) <PRE> 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 </PRE><HR>Jensen's Medium (Sesbania) <PRE> 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 </PRE><HR>Jensen's Medium (1942) <PRE> 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. </PRE><HR>1000X Micronutrients (200 ml) <PRE> H3BO3 572 mg MnCl2 406 mg ZnSO4H2O 44 mg CuSO45 H2O 16 mg NaMO4BH2O 18 mg Co2+ 20 mg Ni2+ 20 mg </PRE><HR>Nodule grinding buffer (500 ml) 50 mM KH2PO4 (pH 7.6), 0.15 M NaCl <PRE> 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. </PRE><HR>Rapidyne germicide <PRE> For working solution: Dilute concentrated stock at 0.6ml/L. </PRE><HR>1 M Tris-Cl (500 ml) <PRE> To 400 ml, add: Tris base 60.5 g Add conc. HCl to pH bring to volume. </PRE><HR>Tris buffer mixtures for pH <PRE> 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 </PRE><HR>Southern Transfer solutions <PRE> 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. </PRE><HR>Random-primer oligonucleotide labelling <PRE> 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. <HR> <H4>MS MEDIUM FOR ARABIDOPSIS</H4> 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) <HR> <H4>B5 MEDIUM FOR ARABIDOPSIS</H4> 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 </PRE><HR>10X Micronutrients (per 100 ml) <PRE> 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) </PRE> <hr> LB (Luria-Bertani) Broth <PRE> 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. </PRE> <hr> CA (Complete Agar) <PRE> Casein Hydrolysate 10 g Yeast Extract 5 g K2HPO4 4 g Agar 15 g H2O up to 1 L </PRE> <hr> Stab Agar <PRE> 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. </PRE> <hr> S.O.C. (from BRL) <PRE> 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. </PRE> <hr> Bacillus Thuringiensis Medium <PRE> 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 -- 121°C </PRE> <HR> </DL>