Team:Goettingen/Project/Materials

From 2012.igem.org

(Difference between revisions)

Latest revision as of 16:15, 26 September 2012


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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-broth

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 x 2 H₂O 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

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

Non-mutated strains

These strains may not contain any plasmids or resistances, because they should be used for transformation or other experiments.

DH10B

Genotype

F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ-

Genetical Characteristics

Antibiotic resistance: none

Reference

Invitrogen

DH5α

Genotype

F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–

Genetical Characteristics

Antibiotic resistance: none

Reference

Invitrogen

BL21DE3

Genotype

E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)

Genetical Characteristics

Antibiotic resistance: none!

Reference

Merck Millipore

XL1 blue

Genotype

endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+)

Genetical Characteristics

Antibiotic resistance: none!

Reference

Merck Millipore

MG1655

Genotype

F-λ-, rph-1

Genetical Characteristics

Antibiotic resistance: none!

Reference

Coli Genetic Stock Center

RP437

Genotype

F-, thr-1, araC14, leuB6(Am), fhuA31, lacY1, tsx-78, λ-, eda-50, hisG4(Oc), rfbC1?, rpsL136(strR), xylA5, mtl-1, metF159(Am), thiE1

Genetical Characteristics

Antibiotic resistance: none!

Reference

Coli Genetic Stock Center

Mutated strains

∆tar

Genotype

F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, Δtar-739::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514

Genetical Characteristics

Antibiotic resistance: Kanamycin

Reference

Coli Genetic Stock Center

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!

LB-agar

LB-agar plates were prepared as follow:

1% agar (w/v) was added to 1 L LB-broth
LB-agar was autoclaved
LB-agar was poured in plates

The E. coli strains were plated out and stored at 4°C. The plates were renewed every two weeks.

↑ Back to top!

sub>2

@@ Line 1: / Line 1: @@
-{{GoettingenHeader}}
+{{GoettingenHeader|deu=Team:Goettingen/Project/Materials|eng=Team:Goettingen/Project/Materials}}
 {{GoettingenBlue}}
 == 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 ====
+==== LB-broth ====
-* 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> x 2 H<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 ===
-Stock solutions and concentrations of antibiotics.
+* Stock solutions and concentrations of antibiotics.
+{| class="goetable"
+|-
+! 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	||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.
+|}
 === 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
+* <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-Benzyl</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>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 46: / Line 58: @@
 ==== 5x M9 salts stock solution ====
 {| class="goetable"
+|-
 !Amount !! Substance
 |-
@@ Line 59: / Line 72: @@
 |-
 |}
-* Stir until dissolved; sterilize by autoclaving at 121°C.
+* Stir until dissolved
 ==== CaCl<sub>2</sub> Buffer for competent cells ====
 {| class="goetable"
-!Amount !! Substance !! Notes
 |-
-| 7.351 g|| CaCl<sub>2</sub> &times; 7 H<sub>2</sub>O || Alternative 0.1 M CaCl<sub>2</sub>.
+!Amount !! Substance !! Notes
 |-
-|15%  || Glycerol ||
+| 7.351 g|| CaCl<sub>2</sub> &times; 7 H<sub>2</sub>O || Alternative 0.1 M CaCl<sub>2</sub>
 |-
-|ad 500 ml || ddH<sub>2</sub>O ||
+|15%  || Glycerol ||
+|-
+|ad 500 ml || ddH<sub>2</sub>O ||
 |-
 |}
@@ Line 76: / Line 90: @@
 ==== 50x TAE for agarose gels ====
 {| class="goetable"
+|-
 ! Amount !! Substance
 |-
 |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> ==
-=== Strains ===
+*<i>E. coli</i> strain list - List of the working <i>E. coli</i> strains including genotypes, genetic markers, and alleles.
-<i>E. coli</i> strain list - List of the working <i>E. coli</i> strains including genotypes, genetic markers, and alleles.
+=== Non-mutated strains ===
+These strains may not contain any plasmids or resistances, because they should be used for transformation or other experiments.
+<br>
+==== DH10B ====
+===== Genotype =====
+*<i>F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ- </i>
+===== Genetical Characteristics =====
+* Antibiotic resistance: none
+===== Reference =====
+* <html><a href="http://products.invitrogen.com/ivgn/product/18297010">Invitrogen</a></html>
+<br>
+==== DH5α ====
+===== Genotype =====
+*<i>F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–</i>
+===== Genetical Characteristics =====
+* Antibiotic resistance: none
+===== Reference =====
+* <html><a href="http://www.invitrogen.com/1/3/dh5a-competent-cells">Invitrogen</a></html>
+<br>
+==== BL21DE3 ====
+===== Genotype =====
+*<i>E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS) </i>
+===== Genetical Characteristics =====
+* Antibiotic resistance: none!
+===== Reference =====
+* <html><a href="http://www.merckmillipore.com/germany/life-science-research/bl21de3-glycerol-stock/EMD_BIO-69387/p_52yb.s1OQ1MAAAEj7Bt9.zLX">Merck Millipore</a></html>
+<br>
+==== XL1 blue ====
+===== Genotype =====
+*<i>endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+) </i>
+===== Genetical Characteristics =====
+* Antibiotic resistance: none!
+===== Reference =====
+* <html><a href="http://www.merckmillipore.com/life-science-research/competent-cells-spotlight-main/c_CzSb.s1O1SAAAAEhSc8sgj.x?back=true&cid=bios-x-goog-1UKEN1277">Merck Millipore</a></html>
+<br>
+==== MG1655 ====
+===== Genotype =====
+*<i>F-λ-, rph-1</i>
+===== Genetical Characteristics =====
+* Antibiotic resistance: none!
+===== Reference =====
+*<html><a href="http://cgsc.biology.yale.edu/Strain.php?ID=4837">Coli Genetic Stock Center</a></html>
+<br>
+==== RP437 ====
+===== Genotype =====
+*<i>F-, thr-1, araC14, leuB6(Am), fhuA31, lacY1, tsx-78, λ-, eda-50, hisG4(Oc), rfbC1?, rpsL136(strR), xylA5, mtl-1, metF159(Am), thiE1</i>
+===== Genetical Characteristics =====
+* Antibiotic resistance: none!
+===== Reference =====
+*<html><a href="http://cgsc.biology.yale.edu/Strain.php?ID=111960">Coli Genetic Stock Center</a></html>
+<br>
+=== Mutated strains ===
+==== <i>∆tar</i> ====
+===== Genotype =====
+*<i>F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, Δtar-739::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514</i>
+===== Genetical Characteristics =====
+* Antibiotic resistance: Kanamycin
+===== Reference =====
+*<html><a href="http://cgsc.biology.yale.edu/Strain.php?ID=107842">Coli Genetic Stock Center</a></html>
 <br>
@@ Line 99: / Line 173: @@
 ==== 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!
+==== LB-agar ====
+LB-agar plates were prepared as follow:
+* 1% agar (w/v) was added to 1 L LB-broth
+* LB-agar was autoclaved
+* LB-agar was poured in plates
+The <i>E. coli</i> strains were plated out and stored at 4°C. The plates were renewed every two weeks.
 {{GoettingenFooter}}
+sub>2

Team:Goettingen/Project/Materials

From 2012.igem.org

Latest revision as of 16:15, 26 September 2012

Contents

Recipes

Media

LB-broth

M9 Swimming Agar

Tryptone swimming agar

Antibiotics

Supplements

Buffer and Solution

5x M9 salts stock solution

CaCl2 Buffer for competent cells

50x TAE for agarose gels

E. coli

Non-mutated strains

DH10B

Genotype

Genetical Characteristics

Reference

DH5α

Genotype

Genetical Characteristics

Reference

BL21DE3

Genotype

Genetical Characteristics

Reference

XL1 blue

Genotype

Genetical Characteristics

Reference

MG1655

Genotype

Genetical Characteristics

Reference

RP437

Genotype

Genetical Characteristics

Reference

Mutated strains

∆tar

Genotype

Genetical Characteristics

Reference

Storage

Glycerol Stock

LB-agar

CaCl₂ Buffer for competent cells