培養基配方
This recipe describes the preparation of 200 ml medium, which can be used for about 4 weeks when kept at 4°C.
Prepare Solutions A – Eitho, flush each w nitrogen t make them anoxicilter-sterilize, f and store at -20°C
Solution A:
L-Alanine 90 mg
L-Arginine 350 mg
L-Asparagine 300 mg
L-Aspartic acid 236 mg
L-Cystein Hcl 700 mg
L-Glutamine 1360 mg
L-Glutamic acid 600 mg
Glycine 150 mg
L-Histidine 1240 mg
L-Isoleucine 262 mg
L-Leucine 262 mg
L-Lysine HCl 364 mg
L-Methionine 300 mg
L-Ornithine HCl 340 mg
L-Phenylalanine 330 mg
L-Proline 230 mg
L-Serine 1050 mg
L-Threonine 240 mg
L-Tryptophan 204 mg
L-Tyrosine 180 mg
L-Valine 234 mg
Distilled Water 100 ml
Solution B:
KCl 1936 mg
MgSO4 x 7H2O 500 mg
NaH2PO4 x H2O 280 mg
NH4Cl 3200 mg
Citric aciddium, triso salt 400 mg
Distilled water 100 ml
Solution C:
Calcium D-(+)-panthothenate 10 mg
Choline chloride 100 mg
Myo-Inositol 100 mg
Thiamine dichloride 10 mg
Thiamin pyrophosphate 10 mg
Coenzym A, sodium salt 2 mg
Flavin adenine dinucleotide (FAD) 2 mg
?-nicotinamide adenine dinucleotide (NAD) 2 mg
2-mercaptoethanesulfonic acid, sodium salt (MES) 20 mg
N-acetylmuramic acid 50 mg
N-acetylglucosamin 400 mg
D-glucuronic acid, sodium salt 1600 mg
D-galacturonic acid 1600 mg
Pyruvic acid, sodium salt 1100 mg
Fumaric acid, disodium salt 1000 mg
Formic acid, sodium salt 600 mg
Distilled water 100 ml
Solution D:
Pyridoxal - HCl 10 mg
Pyridoxal - Phosphat 10 mg
D,L-lactic acid, sodium salt 1120 mg
Hypoxanthine 2.8 mg
Uracil 2.2 mg
Thymidine 4.8 mg
N-(2-acetamido)-2-aminoethanesulfonic acid 3.6 mg
D,L-carnithine 400 mg
Putrescine 2 HCl 10 mg
Phenol red, sodium salt 20 mg
Distilled water 100 ml
Solution E:
D-Glucose 4 g
D-Fructose 1.6 g
D-Maltose 1.6 g
D-Mannitol 1.6 g
D-Mannose 4 g
D-Arabinose 4 g
L-Fucose 4 g
D-Trehalose 4 g
D-Sucrose 4 g
L-Rhamnose 4 g
Distilled water 100 ml
Prepare Solution F, flush with nitrogen to make it anoxic, filter-sterilize and store at 4°C
Solution F:
CaCl2 x H2O 300 mg
Distilled water 100 ml
Prepare Solutions G and H, flush each with nitrogen to make them anoxic, filtersterilize and store at -20°C
Solution G:
Asialofetuin (1 mg/ml) in distilled water
Solution H:
Vitamine B12 5 mg
D-(+)-biotin 5 mg
Folic acid 5 mg
Folinic acid, calcium salt 10 mg
Nicotinamide 5 mg
Nicotinic acid 10 mg
Riboflavine 1 mg
Distilled water 100 ml
Prepare Solution I and store at -20°C
Solution I:
Isobutyric acid 10 μl
2-methylbutyric acid 10 μl
Valeric acid 10 μl
Isovaleric acid 10 μl
Solution J:
D,L-?-lipoic acid (1 mg/ml); in 2-mercaptoethanol-ethanol (1:10); store at -20°C
Solution K:
Hemin (0.2 mM) in NaOH (10 mM)
Solution L:
Homogenize 42 g fresh baker yeast in 140 ml distilled water, incubate for 24h at 56°C
and centrifuge to harvest the supernatant. Adjust pH to 7 with NaOH, make anoxic,
filter-sterilize and store at -80°C
Prepare Trace elements I – III, flush each with nitrogen to make them anoxic, filtersterilize and store at 4°C
Trace elements I
CuSO4 7.89 mg
MnSO4 x H2O 169 mg
ZnSO4 x 7H2O 2.87 g
10 mM HCl 100 ml
Trace elements II
Na2SeO3 173 mg
10mM HCl 100 ml
Trace elements III
NiSO4 x 6H2O 13.1 mg
SnCl2 x 2H2O 11.8 mg
NaVO3 61 mg
(NH4)6Mo7O24 x H2O 1240 mg
10 mM HCl 100 ml
Prepare Supplements 1 - 3, flush each with nitrogen to make them anoxic, filter sterilize and store at -20°C
Supplement 1:
FeSO4 x 7H2O (10 mM) in HCl (10 mM)
Supplement 2:
Ascorbic acid (10%; w/v); in distilled water, neutralize with NaOH
Supplement 3:
Glutathion (0.5 M); in distilled water, neutralize with NaOH
Supplement 4:
Cholesterol (1 mg/ml); in Ethanol (100%); store at -20°C
Supplement 5:
NaHCO3 (10%; w/v) in distilled water, store at 4°C
Preparation of medium
Distilled water 100 ml
Solution A-G 10 ml of each
Solution H 200 μl
Solution I 8 μl
Solution J 2 μl
Solution K 40 μl
Solution L 2 ml
Trace elements I and II pre-dilute 1:1000; use 2 ml of each
Trace elements III pre-dilute 1:1000; use 200 μl
Add distilled water to 200 ml
Supplements 2, 3, 5 2 ml of each
Supplements 1, 4 200 μl of each
Add 10 ml rabbit serum, flush the medium with N2CO2, adjust the pH to 6.9 and filter sterilize the completed medium into tubes.
