Team:Goettingen/notebook materials

General Materials

Antibiotics
Ampicillin 100 µg/mL
Gentamicin 10 µg/mL
Kanamycin 50 µl/mL
Chloramphenicol 15 µg/mL

Supplements
Tryptophane 50 mg/l
Histidine 20 mg/L
Leucine 100 mg/L
Uracile 20 mg/L
Adenine 100 mg/L
3-50 mM 3-amino-1,2,4-triazole (3-AT)

LB medium
For 1 liter:
10 g Tryptone
5 g Yeast Extract
10 g NaCl
add optional:
15 g agar-agar

YPD medium
For 1 liter:
20 g Tryptone
10 g Yeast Extract
20 g Glucose
or after autoclaving 50 mL of 40 % Glucose-solution
add optional:
15 g agar-agar

YPAD medium
prepare YPD medium and add 120 mg adenine.

50 fold ASPA solution
For 1 liter:
297.45 g NaNO3,
26.09 g KCl,
76.21 g KH2PO4,
pH 5.5

1000 fold Trace elements solution
For 1 liter:
12.27 g ZnSO4,
11.01 g H3Bo3,
2.73 g FeSO4,
3.15 g MnCl2,
0.922 g CoCl2,
1.022 g CuSO4,
1.277 g Na2MoO4,
64.77 g Na2EDTA

Minimal medium for Aspergillus cultivation
For 1 liter:
25 mL Glucose 40 %,
1 mL Trace elements 1000x,
20 mL ASPA 50x,
2 mL 1 M MgSO4 


50 fold TAE buffer
For 1 liter:
242 g Tris,
100 mL EDTA (0.5 M; pH 8.0)
dissolve in 60 % of final volume,
add 57.1 mL acetic acid (100 %)
and fill up to 1 liter.


SC dropout medium
6.7 g Yeast Nitrogen Base (without amino acids)
1,46 g Amino acid mixture (-Leu/-Trp/-His/-Ura)
Adjust pH to 5.6
Autoclave 15 min only at 121°C
Add 50 mL sterile Glucose (40%), possible to add 3-50 mL 3-AT

SOB medium
20 g tryptone
5 g Yeast extract
0,584 g NaCl
0,186 g KCl
ad dH2O to 1000 mL
2,032 g MgCl2
2,064 g MgSO4
pH with NaOH to 7.0

Materials & Methods for yeast protocols

AA-mixture
For 1 liter (selection):
Arginine 50 mg,
Aspartic Acid 80 mg,
Histidine 20 mg,
Isoleucine 50 mg,
Leucine 100 mg,
Lysine 50 mg,
Methionine 20 mg,
Phenylalanine 50 mg,
Threonine 100 mg,
Tryptophan 50 mg,
Tyrosine 50 mg,
Uracil 20 mg,
Valine 140 mg,
Serine 20 mg,
Adenine 120 mg,
Histidine 20mg

Synthetic complete dropout (SC/SD) medium
For 1 liter:
6.7 g yeast nitrogen base (YNB) without amino acids,
add optional 18 g Bacto-Agar (autoclave only 15 min),
add. 50 mL of 40 % sterile glucose and recommended amino acids after autoclaving.

SORB
For 1 liter:
6.59 g LiAC,
1.21 g Tris adj. pH with HCl to 8.0;
add 0.37 g 2NaH2EDTA,
182.2 g sorbitol pH 8.0 with acetic acid

LIT-PEG
For 1 liter:
6.59 g LiAC,
1.21 g Tris,
0.37 g 2NaH2EDTA,
400 g PEG 4000 (prepare 10 mL prior to use)

TE buffer
For 1 liter:
0.12 g Tris,
0.37 g 2NaH2EDTA

5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal-stock, 500x)
For 5 mL:
dissolve 100 mg X-α-Gal in dimethylformamide (DMF; toxic).
Store X-α-Gal solutions at -20°C in the dark.

3-amino-1,2,4-triazole (3-AT, 1 M)
For 50 mL:
dissolve 4.2 g 3-AT in sterile water.
Store at 4°C in the dark.

Materials & Methods for E.coli protocols

SOB-medium (super optimal broth) For 1 liter:
5 g yeast extract,
20 g tryptone,
0.584 g NaCl,
0.186 g KCl,
2.4 g MgSO4
Adjust to pH 7.5 prior to use.
This requires approximately 25 ml of 1M NaOH per liter.

TB-buffer
For 1 liter:
18.65 g potassium chloride,
2.2 g calcium chloride,
20 mL 0.5 M PIPES
add 800 mL water
Adjust pH to 6.7 with 1M KOH.
Add gradually 10.88 g manganese chloride to 100 ml water until it is dissolved,
fill up to 1 liter.
Store at 4°C after sterile filtration.

BD-solutions (100 mL each)
BD I: 0.9 g glucose, 2 g lysozyme + 10 μl RNase (10 mg/ml)/ 200 μl BD I
BD II A: 16 g NaOH, 0.315 g EDTA
BD II B: 2 g SDS, 0.3 ml Tris (adjusted with HCl to pH 8 )
→ working solution: mix stock A & B in a 1:1 ratio
BD III: 29.45 g potassium acetate, 5.4 mL formic acid

CCMB80 buffer
For 1 liter:
10 ml of a 1M KOAc stock pH 7.0,
11.8 g CaCl2 ∙ 2 H2O,
4 g MnCl2 ∙ 4 H2O,
2 g MgCl2 ∙ 6 H2O,
100 mL glycerol 100 %
Lower pH to 6.4 with 0.1 M HCl
- An increase in pH will result in precipitation of manganese dioxide from Mn containing solutions,
sterile filtration and store at 4°C.