Team:Goettingen/notebook materials
From 2014.igem.org
General Materials
Antibiotics
Name | Concentration | Provider |
---|---|---|
Ampicillin | 100 µg/ml | |
Gentamicin | 10 µg/ml | |
Kanamycin | 50 µl/ml | |
Chloramphenicol | 15 µg/ml |
Supplements
Name | Concentration | Provider |
---|---|---|
Tryptophane | 50 mg/l | |
Histidine | 20 mg/l | |
Leucine | 100 mg/l | |
Uracile | 20 mg/l | |
Adenine | 100 mg/l | |
3-amino-1,2,4-triazole (3-AT) | 3-50 mM |
LB medium
For 1 liter:
Reagent | Amount | Provider |
---|---|---|
Tryptone | 10 g | |
Yeast Extract | 5 g | |
NaCl | 10 g | |
add if needed: | ||
Agar-agar | 15 g |
YPD medium
For 1 liter:
Reagent | Amount | Provider |
---|---|---|
Tryptone | 20 g | |
Yeast Extract | 10 g | |
Glucose | 20 g or after autoclaving 50 ml of 40 % Glucose-solution | |
add if needed: | ||
Agar-agar | 15 g |
YPAD medium
prepare YPD medium and add 120 mg adenine.
50 fold ASPA solution
For 1 liter:
Reagent | Amount | Provider |
---|---|---|
NaNO3 | 297.45 g | |
KCl | 26.09 g | |
KH2PO4 | 76.21 g | |
pH 5.5 |
1000 fold Trace elements solution
For 1 liter:
Name | Amount | Provider |
---|---|---|
ZnSO4 | 12.27 g | |
H3Bo3 | 11.01 g | |
FeSO4 | 2.73 g | |
MnCl2 | 3.15 g | |
CoCl2 | 0.922 g | |
CuSO4 | 1.022 g | |
Na2MoO4 | 1.277 g | |
Na2EDTA | 64.77 g |
Minimal medium for Aspergillus cultivation
For 1 liter:
Name | Amount | Provider |
---|---|---|
Glucose 40 % | 25 ml | |
Trace elements 1000x | 1 ml | |
ASPA 50x | 20 ml | |
MgSO4 1 M | 2 ml |
50 fold TAE buffer
For 1 liter:
Name | Amount | Provider |
---|---|---|
Tris | 242 g | |
EDTA (0.5 M; pH 8.0) | 100 ml | |
dissolve in 60 % of final volume | ||
add 57.1 mL acetic acid (100 %) and fill up to 1 liter. |
SC dropout medium
SOB medium
For 1 liter:
Name | Amount | Provider | 20 g |
---|---|---|
Yeast extract | 5 g | |
NaCl | 0.584 g | |
KCl | 0.186 g | |
add dH2O to 1000 ml | ||
MgCl2 | 2.032 g | |
MgSO4 | 2.064 g | |
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.
Materials & Methods for Protein Analysis
5 x SDS Loading Dye
For 10 ml:
3 ml Glycerol (100%)
2 ml SDS (20%)
1.6 ml β-Mercaptoethanol (100%)
2 ml H2O
1.4 ml Tris-HCl (pH 7.0)
10 x PAGE buffer, pH 8.3
For 1 liter: 144.13 g glycine, 30.25 g Tris, 10 g SDS
Denaturing Polyacrylamid Gel Electrophoresis (PAGE)
Running gel (12 %, 2 gels) | Stacking gel (5 %, 2 gels) |
---|---|
6.6 ml H2O | 6.8 ml H2O |
8.0 ml Bis-Acrylamid (37, 5:1) | 1.7 ml Bis-Acrylamid (37, 5:1) |
5.0 ml 1.5 M Tris-HCl (pH 8.8) | 1.25 ml 1.5 M Tris-HCl (pH 6.8) |
200 µl SDS (10 %) | 100 µl SDS (10 %) |
200 µl APS (10 %) | 100 µl APS (10 %) |
10 µl TEMED | 10 µl TEMED |
Fixation solution | 10 % Acetic Acid - 45 % MetOH |
Staining solution | 0.5 % Coomassie Brilliant Blue - 10 % Acetic Acid - 45 % MetOH |
Destaining solution | 10 % Acetic acid |
10 x Buffer W
For 1 liter: 6.05 g Tris/HCl pH 8.0, 8.77 g NaCl, 0.37 g Na2 ∙ EDTA
Buffer E
For 100 ml: use 100 ml buffer w and add 0.536 g D-desthiobiotin
Buffer R
For 100 ml: use 100 ml buffer w and add 0,242 g HABA (hydroxy-azophenyl-benzoic acid) pH 8.0
Isopropyl-β-D-thiogalactopyranosid (IPTG; 1M)
For 10 ml 1 M solution, weight 2.383 g IPTG and dissolve in water.
Make aliquots and store at -20°C after sterile filtration.
End concentration for induction is 1 mM.