Team:Goettingen/notebook materials

From 2014.igem.org

Notebook

General Materials

Antibiotics

Name	Concentration
Ampicillin	100 µg/ml
Gentamicin	10 µg/ml
Kanamycin	50 µl/ml
Chloramphenicol	15 µg/ml

Supplements

Name	Concentration
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
Tryptone	10 g
Yeast Extract	5 g
NaCl	10 g
add if needed:
Agar-agar	15 g

YPD medium
For 1 liter:

Reagent	Amount
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
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
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
Glucose 40 %	25 ml
Trace elements 1000x	1 ml
ASPA 50x	20 ml
MgSO₄ 1 M	2 ml

add if needed: Agar-agar15 g

50 fold TAE buffer
For 1 liter:

Name	Amount
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

Name	Amount
Yeast Nitrogen Base (without amino acids)	6.7 g
Amino acid mixture (-Leu/-Trp/-His/-Ura)	1.46 g
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
For 1 liter:

Name	Amount
Tryptone	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):

Name	Amount
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:

Name	Amount
Yeast nitrogen base (YNB) without amino acids	6.7 g
If needed:
Bacto-Agar (autoclave only 15 min)	18 g
add. 50 ml of 40 % sterile glucose and recommended amino acids after autoclaving.

SORB
For 1 liter:

Name	Amount
LiAC	6.59 g
Tris	1.21 g
adj. pH with HCl to 8.0;
add 0.37 g 2NaH2EDTA
Sorbitol	182.2 g
pH 8.0 with acetic acid

LIT-PEG
For 1 liter:

Name	Amount
LiAC	6.59 g
Tris	1.21 g
2NaH2EDTA	0.37 g
PEG 4000 (prepare 10 mL prior to use)	400 g

TE buffer
For 1 liter:

Name	Amount
Tris	0.12 g
2NaH2EDTA	0.37 g

5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal-stock, 500x)
For 5 mL:

Name	Amount
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:

Name	Amount
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:

Name	Amount
yeast extract	5 g
tryptone	20 g
NaCl	0.584 g
KCl	0.186 g
MgSO₄	2.4 g
Adjust to pH 7.5 prior to use.
This requires approximately 25 ml of 1M NaOH per liter.

TB-buffer
For 1 liter:

Name	Amount
potassium chloride	18.65 g
calcium chloride	2.2 g
0.5 M PIPES	20 ml
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:

Name	Amount
glucose	0.9 g
lysozyme	2 g
+ 10 μl RNase (10 mg/ml)/ 200 μl BD I

BD II A:

Name	Amount
NaOH	16 g
EDTA	0.315 g

BD II B:

Name	Amount
SDS	2 g
Tris (adjusted with HCl to pH 8 )	0.3 ml
→ working solution: mix stock A & B in a 1:1 ratio

BD III:

Name	Amount
potassium acetate	29.45 g
formic acid	5.4 mL

CCMB80 buffer
For 1 liter:

Name	Amount
KOAc 1M stock pH 7.0	10 ml
CaCl₂ ∙ 2 H₂O	11.8 g
MnCl₂ ∙ 4 H₂O	4 g
MgCl₂ ∙ 6 H₂O	2 g
glycerol 100 %	100 mL
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:

Name	Amount
Glycerol (100%)	3 ml
SDS (20%)	2 ml
β-Mercaptoethanol (100%)	1.6 ml
H₂O	2 ml
Tris-HCl (pH 7.0)	1.4 ml

10 x PAGE buffer, pH 8.3
For 1 liter:

Name	Amount
glycine	144.13 g
Tris	30.25 g
SDS	10 g

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:

Name	Amount
Tris/HCl pH 8.0	6.05 g
NaCl	8.77 g
Na₂ ∙ EDTA	0.37 g

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.

For in vivo tests

PBS/PBS+:

For 50 mL: 4 g NaCl, 0.1 g KCl, 0.72 g Na2HPO4, 0.12 g KH2PO4
Add optional: 0.0665 g CaCl2 ∙ 2 H2O, 0.05 g MgCl2 ∙ 6 H2O
Adjust the pH to 7.4 (or 7.2) with HCl.

Calcofluor Fluorescent Brightener 28, Stock Solution (10 mg/ml)

Dissolve 10 mg Calcofluor in 1 ml water, store in the dark and at -20°C.

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