Team:Goettingen/notebook materials

From 2014.igem.org

General Materials

Antibiotics

Name Concentration
Ampicillin100 µg/ml
Gentamicin10 µg/ml
Kanamycin50 µl/ml
Chloramphenicol15 µg/ml


Supplements
Name Concentration
Tryptophane50 mg/l
Histidine20 mg/l
Leucine100 mg/l
Uracile20 mg/l
Adenine100 mg/l
3-amino-1,2,4-triazole (3-AT)3-50 mM


LB medium
For 1 liter:
Reagent Amount
Tryptone10 g
Yeast Extract5 g
NaCl10 g
add if needed:
Agar-agar15 g


YPD medium
For 1 liter:
Reagent Amount
Tryptone20 g
Yeast Extract10 g
Glucose20 g or after autoclaving 50 ml of 40 % Glucose-solution
add if needed:
Agar-agar15 g


YPAD medium
prepare YPD medium and add 120 mg adenine.

50 fold ASPA solution
For 1 liter:
Reagent Amount
NaNO3297.45 g
KCl26.09 g
KH2PO476.21 g
pH 5.5


1000 fold Trace elements solution
For 1 liter:
Name Amount
ZnSO412.27 g
H3Bo311.01 g
FeSO42.73 g
MnCl23.15 g
CoCl20.922 g
CuSO41.022 g
Na2MoO41.277 g
Na2EDTA64.77 g


Minimal medium for Aspergillus cultivation
For 1 liter:
Name Amount
Glucose 40 %25 ml
Trace elements 1000x1 ml
ASPA 50x 20 ml
MgSO4 1 M2 ml


add if needed: Agar-agar15 g

50 fold TAE buffer
For 1 liter:
Name Amount
Tris242 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
Tryptone20 g
Yeast extract 5 g
NaCl0.584 g
KCl0.186 g
add dH2O to 1000 ml
MgCl22.032 g
MgSO42.064 g
pH with NaOH to 7.0


Materials & Methods for yeast protocols

AA-mixture
For 1 liter (selection):

Name Amount
Arginine50 mg
Aspartic Acid80 mg
Histidine20 mg
Isoleucine50 mg
Leucine100 mg
Lysine50 mg
Methionine20 mg
Phenylalanine50 mg
Threonine100 mg
Tryptophan50 mg
Tyrosine50 mg
Uracil20 mg
Valine140 mg
Serine20 mg
Adenine120 mg
Histidine20mg


Synthetic complete dropout (SC/SD) medium
For 1 liter:
Name Amount
Yeast nitrogen base (YNB) without amino acids6.7 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
LiAC6.59 g
Tris1.21 g
adj. pH with HCl to 8.0;
add 0.37 g 2NaH2EDTA
Sorbitol182.2 g
pH 8.0 with acetic acid


LIT-PEG
For 1 liter:
Name Amount
LiAC6.59 g
Tris1.21 g
2NaH2EDTA0.37 g
PEG 4000 (prepare 10 mL prior to use)400 g


TE buffer
For 1 liter:
Name Amount
Tris0.12 g
2NaH2EDTA0.37 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 extract5 g
tryptone20 g
NaCl0.584 g
KCl0.186 g
MgSO42.4 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 chloride18.65 g
calcium chloride2.2 g
0.5 M PIPES20 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
glucose0.9 g
lysozyme2 g
+ 10 μl RNase (10 mg/ml)/ 200 μl BD I


BD II A:
Name Amount
NaOH16 g
EDTA 0.315 g


BD II B:
Name Amount
SDS2 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 acetate29.45 g
formic acid5.4 mL


CCMB80 buffer
For 1 liter:
Name Amount
KOAc 1M stock pH 7.010 ml
CaCl2 ∙ 2 H2O11.8 g
MnCl2 ∙ 4 H2O4 g
MgCl2 ∙ 6 H2O2 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
H2O2 ml
Tris-HCl (pH 7.0)1.4 ml


10 x PAGE buffer, pH 8.3
For 1 liter:
Name Amount
glycine144.13 g
Tris30.25 g
SDS10 g



Denaturing Polyacrylamid Gel Electrophoresis (PAGE)

Running gel (12 %, 2 gels)Stacking gel (5 %, 2 gels)
6.6 ml H2O6.8 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 TEMED10 µl TEMED

Fixation solution10 % Acetic Acid - 45 % MetOH
Staining solution0.5 % Coomassie Brilliant Blue - 10 % Acetic Acid - 45 % MetOH
Destaining solution10 % Acetic acid

10 x Buffer W
For 1 liter:

Name Amount
Tris/HCl pH 8.06.05 g
NaCl8.77 g
Na2 ∙ EDTA0.37 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.