Team:Goettingen/Project/Materials

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Recipes

Media

Recipes for liquid and solid media for bacterial cultivation. Stir until dissolved; sterilize by autoclaving 121 °C. Do not forget a magnetic stirrer.

LB-Medium

1 % (w/v) bacto tryptone
0.5 % (w/v) bacto yeast extract
1 % (w/v) NaCl
if required: ad 1 % (w/v) agar

M9 Swimming Agar

1.25 % (v/v) glycerol
20 % (v/v) 5x M9 salt stock solution
0.1 % (v/v) of CaCl₂ - 2H₂O stock solution (20 mg/mL)
0.1 % (v/v) MgSO₄ stock solution (0.12 g/mL)
0.3 % (w/v) of agar

Tryptone swimming agar

1 % (w/v) bacto tryptone
0.5 % (w/v) NaCl
0.3 % (w/v) agar

Antibiotics

Stock solutions and concentrations of antibiotics.

Reagent	Abbreviation	Stock Conc. [mg/mL]	Working Conc. [µg/mL]	Dilution	Solvent	Notes
Ampicillin	Amp	100	100	1,000x	ddH₂O	Culture plates with Amp can be stored at 4 °C for about 2 weeks. Stock solutions can be stored at 4 °C for 2 weeks but can last as long as 4-6 months when stored at -20 °C.
Chloramphenicol	Cm	20	20	1,000x	EtOH
Kanamycin	Kan	50	50	1,000x	ddH2O	Plates may only last a few weeks at 4 °C.

Supplements

Aminoacid mix (containing all aminoacids except tryptophane): stocksolution 0.2 % (w/v), varying amounts used in swimming assays, view methods and notebook of group 1
Caffeine: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
D-Aspartate: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
2-Ethyl-1-Hexanole: stocksolution 5 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
Geraniol: stocksolution 5 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
L-Aspartate-4-Benzylester: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
L-aspartate: stocksolution 10 mM, varying amounts used in swimming assays, view methods and notebook of group 1
Leucin: stocksolution 30.49 mM, add to M9-agar: 4 mL (final concentration 0.305 µM)
Methionin: stocksolution 26.8 mM, add to M9-agar: 4 mL (final concentration 0.268 µM)
Sodium Cyclamate: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
Tryptone: stocksolution 0.5 % (w/v), varying amounts used in swimming assays, view methods and notebook of group 1
Vanillin: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods

Buffer and Solution

Recipes for stocks of buffers and solutions.

5x M9 salts stock solution

Amount	Substance
64 g	Na₂HPO₄ × 7 H₂O
15 g	KH₂PO₄
2.5 g	NaCl
5.0 g	NH₄Cl
ad 1000 ml	ddH₂O

Stir until dissolved

CaCl₂ Buffer for competent cells

Amount	Substance	Notes
7.351 g	CaCl₂ × 7 H₂O	Alternative 0.1 M CaCl₂
15%	Glycerol
ad 500 ml	ddH₂O

Stir until dissolved; sterilize by sterile filtration.

50x TAE for agarose gels

Amount	Substance
242 g	Tris base
57.1 mL	Glacial acetic acid
18.6 g	EDTA
ad 1000 mL	ddH₂O

Stir until dissolved. Working concentration is 1x (40 mL of 50x stock ad 2 L ddH₂O).

E. coli

Strains

E. coli strain list - List of the working E. coli strains including genotypes, genetic markers, and alleles.

Storage

Storage of E. coli strains - How to keep E. coli strains.

Glycerol Stock

The glycerol stock is prepared as follow:

900 µL over night culture in LB-liquid medium
900 µL 50 % sterile glycerol

Glycerol is extremely viscous. Hence, use cut off tips to pipette. Glycerol stocks were vortexed to ensure uniformly mixture and shock frozen in N₂ or dry ice. Then, cultures were stored at -80 °C. Do NOT forget to fill in the according GMO S1 construction sheet!

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sub>2

@@ Line 4: / Line 4: @@
 == Recipes ==
 === Media ===
-Recipes for liquid and solid media for bacterial cultivation. Stir until dissolved; sterilize by autoclaving 121°C. Do not forget a magnetic stirrer.
+Recipes for liquid and solid media for bacterial cultivation. Stir until dissolved; sterilize by autoclaving 121 °C. Do not forget a magnetic stirrer.
 ==== LB-Medium ====
-* 1% (w/v) bacto tryptone
+* 1 % (w/v) bacto tryptone
-* 0.5% (w/v) bacto yeast extract
+* 0.5 % (w/v) bacto yeast extract
-* 1% (w/v) NaCl
+* 1 % (w/v) NaCl
-* if required: ad 1% (w/v) agar
+* if required: ad 1 % (w/v) agar
 ==== M9 Swimming Agar ====
-* 1.25% (v/v) glycerol
+* 1.25 % (v/v) glycerol
-* 20% (v/v) 5x M9 salt stock solution
+* 20 % (v/v) 5x M9 salt stock solution
-* 0.1% (v/v) of CaCl<sub>2</sub> - 2H<sub>2</sub>O stock solution (20 mg/mL)
+* 0.1 % (v/v) of CaCl<sub>2</sub> - 2H<sub>2</sub>O stock solution (20 mg/mL)
-* 0.1% (v/v) MgSO<sub>4</sub> stock solution (0.12 g/mL)
+* 0.1 % (v/v) MgSO<sub>4</sub> stock solution (0.12 g/mL)
 * 0.3 % (w/v) of agar
 ==== Tryptone swimming agar ====
-* 1% (w/v) bacto tryptone
+* 1 % (w/v) bacto tryptone
-* 0.5% (w/v) NaCl
+* 0.5 % (w/v) NaCl
-* 0.3% (w/v) agar
+* 0.3 % (w/v) agar
 === Antibiotics ===
@@ Line 31: / Line 31: @@
 ! Reagent 	!!Abbreviation 	!! Stock Conc. [mg/mL]	!! Working Conc. [µg/mL]!! Dilution 	!! Solvent 	        !!style="width:400px"|Notes
 |-
-|Ampicillin 	||Amp 	||style="text-align:right"| 100	||style="text-align:right"| 100	||style="text-align:right"| 1,000x	||ddH2O 	                ||Culture plates with Amp can be stored at 4°C for about 2 weeks. Stock solutions can be stored at 4°C for 2 weeks but can last as long as 4-6 months when stored at -20°C.
+|Ampicillin 	||Amp 	||style="text-align:right"| 100	||style="text-align:right"| 100	||style="text-align:right"| 1,000x	||ddH<sub>2</sub>O 	                ||Culture plates with Amp can be stored at 4 °C for about 2 weeks. Stock solutions can be stored at 4 °C for 2 weeks but can last as long as 4-6 months when stored at -20 °C.
 |-
 |Chloramphenicol ||Cm 	||style="text-align:right"| 20  	||style="text-align:right"| 20 ||style="text-align:right"| 1,000x 	||EtOH 	                        ||
 |-
-|Kanamycin 	||Kan 	||style="text-align:right"| 50  	||style="text-align:right"| 50 ||style="text-align:right"| 1,000x 	||ddH2O	                ||Plates may only last a few weeks at 4°C.
+|Kanamycin 	||Kan 	||style="text-align:right"| 50  	||style="text-align:right"| 50 ||style="text-align:right"| 1,000x 	||ddH2O	                ||Plates may only last a few weeks at 4 °C.
 |}
 === Supplements ===
-* <u>Aminoacid mix</u> (containing all aminoacids except tryptophane): stocksolution 0.2% (w/v), varying amounts used in swimming assays, view [[Team:Goettingen/Project/Methods|methods]] and notebook of group 1
+* <u>Aminoacid mix</u> (containing all aminoacids except tryptophane): stocksolution 0.2 % (w/v), varying amounts used in swimming assays, view [[Team:Goettingen/Project/Methods|methods]] and notebook of group 1
-* <u>Caffeine</u>: stocksolution 10 mM, 100 µl applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
+* <u>Caffeine</u>: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
-* <u>D-Aspartate</u>: stocksolution 10 mM, 100 µl applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
+* <u>D-Aspartate</u>: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
-* <u>2-Ethyl-1-Hexanole</u>: stocksolution 5 mM, 100 µl applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
+* <u>2-Ethyl-1-Hexanole</u>: stocksolution 5 mM, 100 µL applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
-* <u>Geraniol</u>: stocksolution 5 mM, 100 µl applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
+* <u>Geraniol</u>: stocksolution 5 mM, 100 µL applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
-* <u>L-Aspartate-4-Benzylester</u>: stocksolution 10 mM, 100 µl applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
+* <u>L-Aspartate-4-Benzylester</u>: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
 * <u>L-aspartate</u>: stocksolution 10 mM, varying amounts used in swimming assays, view [[Team:Goettingen/Project/Methods|methods]] and notebook of group 1
-* <u>Leucin</u>: stocksolution 30.49 mM, add to M9-agar: 4 ml (final concentration 0.305 µM)
+* <u>Leucin</u>: stocksolution 30.49 mM, add to M9-agar: 4 mL (final concentration 0.305 µM)
-* <u>Methionin</u>: stocksolution 26.8 mM, add to M9-agar: 4 ml (final concentration 0.268 µM)
+* <u>Methionin</u>: stocksolution 26.8 mM, add to M9-agar: 4 mL (final concentration 0.268 µM)
-* <u>Sodium Cyclamate</u>: stocksolution 10 mM, 100 µl applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
+* <u>Sodium Cyclamate</u>: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
-* <u>Tryptone</u>: stocksolution 0.5% (w/v), varying amounts used in swimming assays, view [[Team:Goettingen/Project/Methods|methods]] and notebook of group 1
+* <u>Tryptone</u>: stocksolution 0.5 % (w/v), varying amounts used in swimming assays, view [[Team:Goettingen/Project/Methods|methods]] and notebook of group 1
-* <u>Vanillin</u>: stocksolution 10 mM, 100 µl applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
+* <u>Vanillin</u>: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view [[Team:Goettingen/Project/Methods|methods]]
 === Buffer and Solution ===
@@ Line 95: / Line 95: @@
 |242 g || Tris base
 |-
-|57.1 ml || Glacial acetic acid
+|57.1 mL || Glacial acetic acid
 |-
 |18.6 g || EDTA
 |-
-|ad 1000 ml || ddH<sub>2</sub>O
+|ad 1000 mL || ddH<sub>2</sub>O
 |-
 |}
-* Stir until dissolved. Working concentration is 1x (40 ml of 50x stock ad 2 l ddH<sub>2</sub>O).
+* Stir until dissolved. Working concentration is 1x (40 mL of 50x stock ad 2 L ddH<sub>2</sub>O).
 == <i>E. coli</i> ==
@@ Line 114: / Line 114: @@
 ==== Glycerol Stock ====
 The glycerol stock is prepared as follow:
-* 900 µl over night culture in LB-liquid medium
+* 900 µL over night culture in LB-liquid medium
-* 900 µl 50% sterile glycerol
+* 900 µL 50 % sterile glycerol
 Glycerol is extremely viscous. Hence, use cut off tips to pipette. Glycerol stocks were vortexed to ensure uniformly mixture and shock
-frozen in N<sub>2</sub> or dry ice. Then, cultures were stored at -80°C. Do NOT forget to fill in the according GMO S1 construction sheet!
+frozen in N<sub>2</sub> or dry ice. Then, cultures were stored at -80 °C. Do NOT forget to fill in the according GMO S1 construction sheet!
 {{GoettingenFooter}}
 sub>2