Team:Goettingen/Project/Materials

From 2012.igem.org

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== Recipes ==
== Recipes ==
=== Media ===  
=== Media ===  
-
Recipes for liquid and solid media for bacterial cultivation.
+
Recipes for liquid and solid media for bacterial cultivation. Stir until dissolved; sterilize by autoclaving 121 °C. Do not forget a magnetic stirrer.
-
<html>
+
-
<ol>
+
-
<li><u>LB-Medium</u> </li>
+
-
<div style="text-indent:20px;">1% (w/v) bacto tryptone </div>
+
-
<div style="text-indent:20px;">0.5% (w/v) bacto yeast extract </div>
+
-
<div style="text-indent:20px;">1% (w/v) NaCl </div>
+
-
<div style="text-indent:20px;">if required: ad 1% (w/v) agar </div>
+
-
<br>
+
-
<li><u>M9 Swimming Agar</u> </li>
+
==== LB-broth ====
-
<div style="text-indent:20px;">1.25% (v/v) glycerol </div>
+
* 1 % (w/v) bacto tryptone
-
<div style="text-indent:20px;">20% (v/v) 5x M9 salt stock solution </div>
+
* 0.5 % (w/v) bacto yeast extract
-
<div style="text-indent:20px;">0.1% (v/v) of CaCl<sub>2</sub> - 2H<sub>2</sub>O stock solution (20 mg/mL)  </div>
+
* 1 % (w/v) NaCl
-
<div style="text-indent:20px;">0.1% (v/v) MgSO<sub>4</sub> stock solution (0.12 g/mL)  </div>
+
* if required: ad 1 % (w/v) agar
-
<div style="text-indent:20px;">0.3 % (w/v) of agar </div>
+
-
<br>
+
-
<li><u>Tryptone swimming agar</u> </li>
+
==== M9 Swimming Agar ====
-
<div style="text-indent:20px;">1% (w/v) bacto tryptone </div>
+
* 1.25 % (v/v) glycerol
-
<div style="text-indent:20px;">0.5% (w/v) NaCl </div>
+
* 20 % (v/v) 5x M9 salt stock solution
-
<div style="text-indent:20px;">0.3% (w/v) agar </div>
+
* 0.1 % (v/v) of CaCl<sub>2</sub> x 2 H<sub>2</sub>O stock solution (20 mg/mL) 
-
<br>
+
* 0.1 % (v/v) MgSO<sub>4</sub> stock solution (0.12 g/mL) 
 +
* 0.3 % (w/v) of agar
-
<br>
+
==== Tryptone swimming agar ====
-
</ol>
+
* 1 % (w/v) bacto tryptone
-
</html>
+
* 0.5 % (w/v) NaCl
 +
* 0.3 % (w/v) agar
=== Antibiotics ===
=== 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 ===
=== 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 ===
=== Buffer and Solution ===
Recipes for stocks of buffers and solutions.
Recipes for stocks of buffers and solutions.
-
# <b><u>5x M9 salts stock solution</u></b><br>
+
==== 5x M9 salts stock solution ====
-
Stir until dissolved; sterilize by autoclaving at 121°C. <br>
+
{| class="goetable"
-
<html><span style="text-indent:20px;"><table width="50%" border="1" bordercolor="#000000" cellpadding="0" cellspacing="2">
+
|-
-
<tr>
+
!Amount !! Substance
-
  <th scope="col">Amount   </th>
+
|-
-
  <th scope="col">Substance</th>
+
|64 g || Na<sub>2</sub>HPO<sub>4</sub> &times; 7 H<sub>2</sub>O
-
</tr>
+
|-
-
<tr>
+
|15 g || KH<sub>2</sub>PO<sub>4</sub>
-
  <td>64 g</td>
+
|-
-
  <td>Na<sub>2</sub>HPO<sub>4</sub> &times; 7 H<sub>2</sub>O</td>
+
|2.5 g || NaCl
-
</tr>
+
|-
-
<tr>
+
|5.0 g || NH<sub>4</sub>Cl
-
  <td>15 g </td>
+
|-
-
  <td>KH<sub>2</sub>PO<sub>4</sub> </td>
+
|ad 1000 ml || ddH<sub>2</sub>O
-
</tr>
+
|-
-
<tr>
+
|}
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  <td>2.5 g</td>
+
* Stir until dissolved
-
  <td>NaCl</td>
+
 
-
</tr>
+
==== CaCl<sub>2</sub> Buffer for competent cells ====
-
<tr>
+
{| class="goetable"
-
  <td>5.0 g</td>
+
|-
-
  <td>NH<sub>4</sub>Cl</td>
+
!Amount !! Substance !! Notes  
-
</tr>
+
|-
-
<tr>
+
| 7.351 g|| CaCl<sub>2</sub> &times; 7 H<sub>2</sub>O || Alternative 0.1 M CaCl<sub>2</sub>
-
  <td>ad 1000 ml</td>
+
|-
-
  <td>ddH<sub>2</sub>O </td>
+
|15% || Glycerol ||
-
</tr>
+
|-
-
</table></span></html>
+
|ad 500 ml || ddH<sub>2</sub>O ||
-
# <b><u>CaCl<sub>2</sub> Buffer for competent cells</u></b><br>
+
|-
-
Stir until dissolved; sterilize by sterile filtration.
+
|}
-
<html><span style="text-indent:20px;"><table width="50%" border="1" bordercolor="#000000" cellpadding="0" cellspacing="2">
+
* Stir until dissolved; sterilize by sterile filtration.
-
<tr>
+
 
-
  <th scope="col">Amount</th>
+
==== 50x TAE for agarose gels ====
-
  <th scope="col">Substance</th>
+
{| class="goetable"
-
  <th scope="col">Notes</th>
+
|-
-
</tr>
+
! Amount !! Substance
-
<tr>
+
|-
-
  <td>7.351 g </td>
+
|242 g || Tris base
-
  <td>CaCl<sub>2</sub> &times; 7 H<sub>2</sub>O</td>
+
|-
-
  <td>Alternative 0.1 M CaCl<sub>2</sub>.</td>
+
|57.1 mL || Glacial acetic acid
-
</tr>
+
|-
-
<tr>
+
|18.6 g || EDTA
-
  <td>15%         </td>
+
|-
-
  <td> Glycerol</td>
+
|ad 1000 mL || ddH<sub>2</sub>O
-
  <td></td>
+
|-
-
</tr>
+
|}
-
  <tr>
+
* Stir until dissolved. Working concentration is 1x (40 mL of 50x stock ad 2 L ddH<sub>2</sub>O).
-
  <td>ad 500 ml</td>
+
-
  <td>ddH<sub>2</sub>O</td>
+
-
  <td></td>
+
-
</tr>
+
-
</table></span></html>
+
-
# <b><u> 50x TAE for agarose gels</u></b><br>
+
-
Stir until dissolved. Working concentration is 1x (40 ml of 50x stock ad 2 l ddH<sub>2</sub>O).
+
-
<html><span style="text-indent:20px;"><table width="50%" border="1" bordercolor="#000000" cellpadding="0" cellspacing="2">
+
-
<tr>
+
-
  <th scope="col">Amount</th>
+
-
  <th scope="col">Substance</th>
+
-
</tr>
+
-
<tr>
+
-
  <td>242 g</td>
+
-
  <td>Tris base</td>
+
-
</tr>
+
-
<tr>
+
-
  <td>57.1 ml        </td>
+
-
  <td>Glacial acetic acid </td>
+
-
</tr>
+
-
  <tr>
+
-
  <td>18.6 g</td>
+
-
  <td>EDTA </td>
+
-
</tr>
+
-
  <tr>
+
-
  <td>ad 1000 ml  </td>
+
-
  <td>ddH<sub>2</sub>O </td>
+
-
</tr>
+
-
</table></span></html>
+
== <i>E. coli</i> ==
== <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>
=== Storage ===
=== Storage ===
Storage of <i>E. coli</i> strains - How to keep <i>E. coli</i> strains.
Storage of <i>E. coli</i> strains - How to keep <i>E. coli</i> strains.
-
* Glycerol Stock
+
==== Glycerol Stock ====
-
** 900 µl over night culture in LB-liquid medium
+
The glycerol stock is prepared as follow:
-
** 900 µl 50% sterile glycerol
+
* 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
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}}
{{GoettingenFooter}}
 +
sub>2

Latest revision as of 16:15, 26 September 2012

Deutsch  / English 

Contents

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


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


BL21DE3

Genotype
  • E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)
Genetical Characteristics
  • Antibiotic resistance: none!
Reference


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


MG1655

Genotype
  • F-λ-, rph-1
Genetical Characteristics
  • Antibiotic resistance: none!
Reference


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


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


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.

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