Team:Goettingen/notebook materials
From 2014.igem.org
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<h2>Materials & Methods for yeast protocols</h2> | <h2>Materials & Methods for yeast protocols</h2> | ||
<p><b>AA-mixture</b><br /> | <p><b>AA-mixture</b><br /> | ||
- | For 1 liter (selection): <br />Arginine 50 mg | + | For 1 liter (selection): <br /> |
+ | <center><table class="table1" style="width:700px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | <th style="font-size:80%;">Provider</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr><td>Arginine</td><td>50 mg</td><td></td></tr> | ||
+ | <tr><td>Aspartic Acid</td><td>80 mg</td><td></td></tr> | ||
+ | <tr><td>Histidine</td><td>20 mg</td><td></td></tr> | ||
+ | <tr><td>Isoleucine</td><td>50 mg</td><td></td></tr> | ||
+ | <tr><td>Leucine</td><td>100 mg</td><td></td></tr> | ||
+ | <tr><td>Lysine</td><td>50 mg</td><td></td></tr> | ||
+ | <tr><td>Methionine</td><td>20 mg</td><td></td></tr> | ||
+ | <tr><td>Phenylalanine</td><td>50 mg</td><td></td></tr> | ||
+ | <tr><td>Threonine</td><td>100 mg</td><td></td></tr> | ||
+ | <tr><td>Tryptophan</td><td>50 mg</td><td></td></tr> | ||
+ | <tr><td>Tyrosine</td><td>50 mg</td><td></td></tr> | ||
+ | <tr><td>Uracil</td><td>20 mg</td><td></td></tr> | ||
+ | <tr><td>Valine</td><td>140 mg</td><td></td></tr> | ||
+ | <tr><td>Serine</td><td>20 mg</td><td></td></tr> | ||
+ | <tr><td>Adenine</td><td>120 mg</td><td></td></tr> | ||
+ | <tr><td>Histidine</td><td>20mg</td><td></td></tr> | ||
+ | </table></center> | ||
<b>Synthetic complete dropout (SC/SD) medium</b><br /> | <b>Synthetic complete dropout (SC/SD) medium</b><br /> |
Revision as of 19:18, 16 October 2014
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
Name | Amount | Provider |
---|---|---|
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 | 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):
Name | Amount | Provider |
---|---|---|
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 |
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.