常用试剂和培养基.docx

1、常用试剂和培养基常用试剂和培养基Sterile water bottles 200 ml, 500 ml, or 950 ml water.Autoclave.0.05 M CaCl2 (per 200 ml) CaCl22 H2O 1.5 gAdd 198 ml water.Autoclave.0.5 M CaCl2 (per 200 ml) CaCl22 H2O 15.0 gAdd 188 ml water.Autoclave.0.1 M MgSO4 (per 200 ml) MgSO4 (anhydrous) 2.4 gAdd 200 ml water.Autoclave.10% MSG

2、 (for 200 ml) Glutamic acid (Na) 20.0 gAdd 188 ml water.Autoclave.40% Glucose (per 200 ml) Glucose(dextrose) 80.0 gAdd water to 200 ml(145-150 ml).Autoclave.10% CAA (per 200 ml) Casein Acid Hydrolysate 20.0 gAdd 186 ml water.Autoclave.10% Yeast (per 200 ml) Yeast extract 20.0 gAdd 150-160 ml water.

3、Autoclave.Add 150 ul -mercaptoethanolafter autoclaving.20X Tryptone/NaCl (per 200 ml) Tryptone 40.0 gNaCl 20.0 gAdd 190 ml water. Autoclave.Add 150 ul -mercaptoethanolafter autoclaving.10X M9 stock (for 200 ml) Na2HPO4 (anhydr.) 12.0 gKH2PO4 6.0 gNaCl 1.0 gNH4Cl 2.0 gAdd 196 ml H2O. Autoclave.Note:

4、leave out NH4Cl to prepare 10X M9-N.10X M9 - N stock (for 200 ml) Na2HPO4 (anhydr.) 12.0 gKH2PO4 6.0 gNaCl 1.0 gAdd 196 ml H2O. Autoclave.Note: leave out NH4Cl!100X P (for 200 ml) KH2PO4 18.1 gK2HPO4 5.0 gpH to 6.3 with 10 N KOH!Add 192 ml H20. Autoclave.1X SM (for 200 ml) NaCl 1.16 gMgSO4 0.20 gGel

5、atin (2%) 1.00 ml or 20 mgAdd 190 ml H2O, then add:Tris (1 M, pH 7.5) 10.0 mlAutoclave.2% Gelatin (per 500 ml) Gelatin 10 gAdd 500 ml water.Autoclave.20% Succinate (for 500 ml) Succinic acid 100 gKOH 90 gAdd to 400 ml ice cold water.Measure pH to 6.4, filter sterilize.10X TBE (electrophoresis buffer

6、) Tris base 108.0 gNa2EDTA 9.3 gBoric Acid 55.0 gBring 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 gAdd 190 ml H2O.Autoclave, store in 60C water bath.R agar stock 200 ml 500 mlTryptone. 2.0 g 5.0

7、gNaCl. 1.0 g 2.5 gAgar. 3.0 g 7.5 gAdd H2O. 194.0 ml 485.0 mlAutoclave, store in 60C water bath.LB agar 200 ml 1000 ml Tryptone . 2 g 10 gNaCl . 1 g 5 gYeast extract . 1 g 5 gAgar . 3 g 15 gAdd H2O . 193 ml 990 mlAutoclave, store in 60C water bath. LB agar can also be made byadding 10 ml 10% yeast e

8、xtract and 10 ml 20X Tryptone/NaCl to190 ml WA.NZ 200 ml 500 ml NZ amine . 2.0 g 5.0 gNaCl . 1.0 g 2.5 gMgSO4 (anhydrous). 0.2 g 0.5 gAdd 10 M NaOH (1 drop/100 ml) after adding water.Autoclave.For agar media:Agar . 2.4 g 6.0 gStir before and after autoclaving.GYPC to WA stock: 200 ml 1000 ml Glucose

9、 (40%) . 2.0 ml 10.0 mlYeast extract (10%) 4.0 ml 20.0 ml100X P . 2.0 ml 10.0 mlCAA (10%) . 4.0 ml 20.0 mlSuccinate (20%). 1.0 ml 5.0 mlAdd appropriate antibiotics.Pour plates.ORS minimal 200 ml 1000 ml 10X M9 . 10 ml 50 mlSuccinate (20%). 2 ml 10 mlMSG (10%) . 2 ml 10 mlGlucose (40%) . 1 ml 5 mlMgS

10、O4 (0.1 M) . 1 ml 5 mlCaCl2 (0.5 M) . 200 ul 1 ml1000X vitamins . 200 ul 1 mlWater . 184 ml 920 mlOmit succinate and add 10 ml glucose for storage medium.E. coli minimal 200 ml 1000 ml 10X M9 . 20 ml 100 mlMgSO4 (0.1 M) . 2 ml 10 mlCaCl2 (0.5 M) . 0.2 ml 1 ml1000X vitamins . 0.2 ml 1 mlGlucose (40%)

11、 . 2 ml 10 mlXC minimal 200 ml 1000 ml 10X M9 . 5.0 ml 25 mlMgSO4 (0.1 M) . 1.0 ml 5 mlCaCl2 (0.5 M) . 0.2 ml 1 ml200X 7 aa stock. 0.2 ml 1 mlGlucose (40%) . 0.5 ml 2.5 mlGlycerol (50%) . 4.0 ml 20 mlNif plates 10X Nif buffer 100 ml10X trace elements 100 ml1000X vitamins 1 mlSterile dd H2O 799 mlAga

12、rose 6 gSuccinate (20%) 20 ml(Succinate is already 0.4% from that in 10X nif buffer)10X Nif buffer 1000 ml K2HPO4 34.02 gKH2PO4 57.06 gSuccinate (20%) 200.00 mlpH should be 6.4 after 10X dilution.1000X ORS vitamins Nicotinic acid 2.0 mg/mlPantothenate 1.0 mg/mlBiotin 0.2 mg/mlStir, filter sterilize.

13、Trace elements MgSO4 (0.1 M) 25 mlCaCl2 (0.5 M) 5 mlNa2MoO4 (1 mg/ml) 5 mlFeCl3 (1 mg/ml) 5 mlWater 460 mlYEM (per 500 ml) K2HPO4 0.5 gMgSO47 H2O 0.2 gNaCl 0.1 gMannitol 10.0 gYeast extract 0.4 gAgar 15.0 gpH to 6.9YEM substitute (per 200 ml) To 180 ml WA stock:10X M9 8.0 mlMannitol (20%) 20.0 mlYea

14、st extract (10%) 1.6 mlSM10 selective media (for storage) 10X versatile Mg 100 mlThiamine 5 mlThreonine 5 mlLeucine 5 mlGlucose (40%) 10 mlWater 875 mlAmino 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 mlTh

15、iamine (1 mg/ml) 2.0 mlCaCl2 0.2 ml200X PLT (for 200 ml) Proline 9.20 gLeucine 1.60 gThiamine 0.67 gAutoclave.Mg PLT 10X versatile Mg 100 ml200X PLT 5 ml40% glucose 10 mlWater 885 ml Z buffer (lacZ assays) 200 ml 1000 mlNa2HPO47 H2O . 3.22 g 16.10 gNaH2PO4H2O . 1.10 g 5.50 gKCl . 0.15 g 0.75 gMgSO4

16、(0.1 M) . 1.00 ml 0.20 ml-mercaptoethanol. 2.70 ml 0.54 mlFilter sterilize.ONPG stock 4 mg/ml in phosphate buffer (pH 7)Phosphate buffer (pH 7.0) per 100 ml: Na2HPO47 H2O 1.64 gNaH2PO4H2O 1.38 g0.2 M Na2CO3 (100 ml) Na2CO3 (anhydr.) 10.6 gJensens Medium (Sesbania) 10X Macro nutrients (1000 ml): K2HP

17、O43 H2O 2.6 g NaCl 2.0 g MgSO4 0.97 g CaCl2 10.0 g FeCl36 H2O 1.4 g KNO3 3.0 gJensens 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 gJensens Medium (1942) see Vincent 1970 p. 75.pe

18、r 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 gDilute 1:5 when used.15 ml diluted medium/pouch.1000X Micronutrients (200 ml) H3BO3 572 mgMnCl2 406 mgZnSO4H2O 44 mgCuSO45 H2O 16 mgNaMO4BH2O 18 mgCo2+ 20 mgNi2+ 20 mgNodule grinding buffer (500 ml)

19、50 mM KH2PO4 (pH 7.6), 0.15 M NaCl K2HPO4 (1M) 3.25 mlKH2PO4 (1M) 21.75 mlNaCl 4.38 gmAdd 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 gAdd conc. HCl t

20、o pHbring to volume.Tris buffer mixtures for pH pH Tris HCl Tris base7.0 .923 .0777.1 .905 .0957.2 .90 .107.3 .865 .1357.4 .84 .167.5 .82 .187.6 .78 .227.7 .74 .267.8 .67 .337.9 .63 .378.0 .50 .50Southern Transfer solutions I. 0.25 N HCl: 21.5 ml conc. HCl (11.6N) to 1 liter volume.II. 0.5 M NaOH, 1

21、.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 oligon

22、ucleotide labelling Mix: 9.00 ul DNA (not more than 200 ng) 1.25 ul hexamersBoil, 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 -mercaptoethanol 1.0 ul BSA (40 mg/ml) 0.5 ul Klenow 5.0 ul -32P CTPLet reaction go at room temperature for

23、at least 4 hours, or overnight.MS MEDIUM FOR ARABIDOPSISTo 990 ml H2O add:Sucrose . 10.0 gMOPS . 0.5 gAgar . 8.0 gAdjust 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)Mg

24、SO47H2O. 15.0 ml 100 mM (24.65 g/L)CaCl27H2O. 10.0 ml 300 mM (44.00 g/L)Glycine. 2.67 ml 10 mMNicotinate. 0.4 ml 10 mMThiamin. 0.02 ml 10 mMPyridoxineHCl. 0.24 ml 10 mMMyoinositol. 5.5 ml 100 mMFeSO47H2O . 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

25、 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 ARABIDOPSISTo 990 ml H2O add:Sucrose .

26、 20.0 gMOPS . 0.5 gAgar . 8.0 gAdjust 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 mMMgSO47H2O. 10.0 ml 100 mM (24.65 g/L)(NH4)2SO3. 10.0 ml 100 mMCaCl27H2O. 3.3 ml 300 mM (44.00 g/L)Myoinositol. 5.5 ml 100 mMThia

27、mine. 3.0 ml 10 mMNicotinate. 0.8 ml 10 mMPyridoxineHCl. 0.48 ml 10 mM10X 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 mlKinetin. 0.05 mlFor Shoot Inducing Medium (SIM) add:IAA. 0.15 ml6-dimethylaminopurine. 5.00 ml10X

28、 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-Trypt