Team:Aachen/Notebook/Protocols/Culture medium and conditions

From 2014.igem.org

(Difference between revisions)
(LB Medium)
(References)
 
(21 intermediate revisions not shown)
Line 18: Line 18:
   <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
   <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
     <a class="menulink" href="http://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black">
     <a class="menulink" href="http://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Culture Media &<br/>Culture Conditions</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Culture Media</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(http://2014.igem.org/wiki/images/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(http://2014.igem.org/wiki/images/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>
Line 44: Line 44:
</html>
</html>
-
= Culture Medium and Culture Conditions =
+
= Culture Media =
-
== Media ==
+
In our project we used different kinds of media for cultivation, transformation and chip preparation. While complex media such as LB offered an easy way of cultivation, minimal media such as M9 or HM provide a low autofluorescence for fluorescence measurements. All used media are listed below.
 +
 
 +
== Complex media ==
=== Luria-Bertani Medium (LB)===
=== Luria-Bertani Medium (LB)===
# weight components
# weight components
Line 64: Line 66:
## '''wait until the pressure valve retracts''' (30-45 minutes)
## '''wait until the pressure valve retracts''' (30-45 minutes)
## open, close caps & shake
## open, close caps & shake
-
# for plates, wait until you can touch the bottle ('''<60&nbsp;°C''', clean bench!)
+
# for plates, wait until you can touch the bottle ('''<60°C''', clean bench!)
-
# add antibiotics (1&nbsp;µL/mL) and '''shake''' (gloves!)
+
# add antibiotics (1&nbsp;µl/ml) and '''shake''' (gloves!)
-
=== TB Medium ===
+
=== Terrific-Broth-Medium (TB) ===
-
# components 1:
+
# components for 1&nbsp;:
-
#: '''4&nbsp;mL/L glycerol'''
+
#: '''4&nbsp;ml/L glycerol'''
#: '''12&nbsp;g/L tryptone'''
#: '''12&nbsp;g/L tryptone'''
#: '''24&nbsp;g/L yeast extract'''  
#: '''24&nbsp;g/L yeast extract'''  
-
# fill up to 900&nbsp;mL with deionized water
+
# fill up to 900&nbsp;ml with deionized water
# '''mix well''' by shaking
# '''mix well''' by shaking
# autoclave
# autoclave
Line 78: Line 80:
#: '''0.17&nbsp;M KH<sub>2</sub>PO<sub>4</sub>'''
#: '''0.17&nbsp;M KH<sub>2</sub>PO<sub>4</sub>'''
#: '''0.72&nbsp;M K<sub>2</sub>HPO<sub>4</sub>'''
#: '''0.72&nbsp;M K<sub>2</sub>HPO<sub>4</sub>'''
-
# dissolve in 100&nbsp;mL deionized water and sterilize it by passing it through a filter
+
# dissolve in 100&nbsp;ml deionized water and sterilize it by passing it through a filter
# after autoclaving and cooling down, add sterile phosphate solutions
# after autoclaving and cooling down, add sterile phosphate solutions
 +
=== Nutrient Agar medium (NA) ===
 +
#Components for 1&nbsp;L
 +
#: '''Enzymatic Digest of Gelatin 5&nbsp;g'''
 +
#: '''Beef Extract 3&nbsp;g'''
 +
#: '''Agar 15&nbsp;g'''
 +
# Final pH: 6.8 ± 0.2 at 25°C
 +
# Suspend 23&nbsp;g of the medium in one liter of purified water.
 +
# Heat with frequent agitation and boil for one minute to completely dissolve the medium.
 +
# Autoclave at 121°C for 15&nbsp;min.
 +
== Minimal Media ==
=== Hartmans Minimal Medium (HM) ===
=== Hartmans Minimal Medium (HM) ===
Line 88: Line 100:
Three '''100x stock solutions''' are prepared according to the following recipe and stored at 4°C:
Three '''100x stock solutions''' are prepared according to the following recipe and stored at 4°C:
-
* '''100x Buffer''',composed of 388&nbsp;g dipotassium phosphate, 212g monosodium phosphate dihydrate per Liter, adjusted to a pH of 7.0 and sterilized by autoclaving.
+
* '''100x Buffer''',composed of 388&nbsp;g dipotassium phosphate, 212&nbsp;g monosodium phosphate dihydrate per Liter, adjusted to a pH of 7.0 and sterilized by autoclaving.
* '''100x ammonium sulfate''' composed of 200&nbsp;g/l ammonium sulfate and sterilized by autoclaving.
* '''100x ammonium sulfate''' composed of 200&nbsp;g/l ammonium sulfate and sterilized by autoclaving.
* '''100x MM salts'''
* '''100x MM salts'''
# Add 1&nbsp;g EDTA to 25&nbsp;ml water.
# Add 1&nbsp;g EDTA to 25&nbsp;ml water.
-
# Add drops of 10 M sodium hydroxide until EDTA is completely dissolved.
+
# Add drops of 10&nbsp;M sodium hydroxide until EDTA is completely dissolved.
# Adjust pH back to 4.0 with concentrated hypochloric acid.
# Adjust pH back to 4.0 with concentrated hypochloric acid.
# Fill up with water to 800&nbsp;ml and dissolve following components:
# Fill up with water to 800&nbsp;ml and dissolve following components:
Line 111: Line 123:
! '''Components for 1&nbsp;L''' !! '''Volume'''
! '''Components for 1&nbsp;L''' !! '''Volume'''
|-
|-
-
| bidest. water || style="text-align:right"| 778.667&nbsp;mL
+
| bidest. water || style="text-align:right"| 778.667&nbsp;ml
|-
|-
-
| 10x Salt solution || style="text-align:right"| 100&nbsp;mL
+
| 10x Salt solution || style="text-align:right"| 100&nbsp;ml
|-
|-
-
| Magnesiumsulfatehaptahydrate (10&nbsp;mM) || style="text-align:right"| 100&nbsp;mL
+
| Magnesiumsulfatehaptahydrate (10&nbsp;mM) || style="text-align:right"| 100&nbsp;ml
|-
|-
-
| Glucose 20% (w/v) || style="text-align:right"| 20&nbsp;mL
+
| Glucose 20% (w/v) || style="text-align:right"| 20&nbsp;ml
|-
|-
-
| 1000x Trace elements || style="text-align:right"| 1&nbsp;mL
+
| 1000x Trace elements || style="text-align:right"| 1&nbsp;ml
|-
|-
-
| Thiamin (1&nbsp;mM) || style="text-align:right"| 0.333&nbsp;mL
+
| Thiamin (1&nbsp;mM) || style="text-align:right"| 0.333&nbsp;ml
|}
|}
Line 167: Line 179:
| Boric acid || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 124&nbsp;mg/L  
| Boric acid || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 124&nbsp;mg/L  
|-
|-
-
| Hydrochloric acid || style="text-align:right"| 1&nbsp;mM || style="text-align:right"| 20&nbsp;mL
+
| Hydrochloric acid || style="text-align:right"| 1&nbsp;mM || style="text-align:right"| 20&nbsp;ml
|-
|-
|}
|}
</center>
</center>
-
=== Nutrient Agar medium (NA) ===
+
== Transformation Medium ==
-
 
+
=== Super Optimal broth with Catabolite repression medium (SOC) ===
=== Super Optimal broth with Catabolite repression medium (SOC) ===
# components
# components
-
#: '''0,5&nbsp;% yeast extract'''
+
#: '''0,5% yeast extract'''
-
#: '''2&nbsp;% tryptone'''
+
#: '''2% tryptone'''
#: '''10&nbsp;mM NaCl'''
#: '''10&nbsp;mM NaCl'''
#: '''2.5&nbsp;mM KCl'''
#: '''2.5&nbsp;mM KCl'''
Line 185: Line 196:
# after autoclaving, add 20&nbsp;mM sterile glucose solution (filter sterilization)
# after autoclaving, add 20&nbsp;mM sterile glucose solution (filter sterilization)
 +
==References==
 +
* Hartmans, S., Smiths J.P., Volkering F., and de Brondth, J.A. (1989). Metabolism of Styrene Oxide and 2-Phenylethanol in the Styrene-Degrading Xanthobacter Strain 124X. Applied and Environmental Microbiology, Nov. Available at: http://www.ncbi.nlm.nih.gov/pubmed/?term=PMC203180.
<html>
<html>
Line 200: Line 213:
   <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
   <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
     <a class="menulink" href="http://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black">
     <a class="menulink" href="http://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Culture Media &<br/>Culture Conditions</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Culture Media</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(http://2014.igem.org/wiki/images/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(http://2014.igem.org/wiki/images/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>

Latest revision as of 03:42, 18 October 2014

Culture Media

In our project we used different kinds of media for cultivation, transformation and chip preparation. While complex media such as LB offered an easy way of cultivation, minimal media such as M9 or HM provide a low autofluorescence for fluorescence measurements. All used media are listed below.

Complex media

Luria-Bertani Medium (LB)

  1. weight components
    5 g/L NaCl
    10 g/L tryptone
    5 g/L yeast extract
    (15 g/L agar for plates)
  2. fill up to 1 L with deionized water
  3. mix well by shaking
  4. autoclave
    1. autoclaving tape, caps slightly unscrewed
    2. base of the pot has to be covered with deionized water
    3. close lid
    4. heat level 3 until the pressure valve opens
    5. reduce heat level to 1.5
    6. set timer to 20 minutes
    7. turn heater off
    8. wait until the pressure valve retracts (30-45 minutes)
    9. open, close caps & shake
  5. for plates, wait until you can touch the bottle (<60°C, clean bench!)
  6. add antibiotics (1 µl/ml) and shake (gloves!)

Terrific-Broth-Medium (TB)

  1. components for 1 :
    4 ml/L glycerol
    12 g/L tryptone
    24 g/L yeast extract
  2. fill up to 900 ml with deionized water
  3. mix well by shaking
  4. autoclave
  5. components 2:
    0.17 M KH2PO4
    0.72 M K2HPO4
  6. dissolve in 100 ml deionized water and sterilize it by passing it through a filter
  7. after autoclaving and cooling down, add sterile phosphate solutions

Nutrient Agar medium (NA)

  1. Components for 1 L
    Enzymatic Digest of Gelatin 5 g
    Beef Extract 3 g
    Agar 15 g
  2. Final pH: 6.8 ± 0.2 at 25°C
  3. Suspend 23 g of the medium in one liter of purified water.
  4. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
  5. Autoclave at 121°C for 15 min.

Minimal Media

Hartmans Minimal Medium (HM)

This mineral salts medium is based on (Hartmans et al., 1989).

Three 100x stock solutions are prepared according to the following recipe and stored at 4°C:

  • 100x Buffer,composed of 388 g dipotassium phosphate, 212 g monosodium phosphate dihydrate per Liter, adjusted to a pH of 7.0 and sterilized by autoclaving.
  • 100x ammonium sulfate composed of 200 g/l ammonium sulfate and sterilized by autoclaving.
  • 100x MM salts
  1. Add 1 g EDTA to 25 ml water.
  2. Add drops of 10 M sodium hydroxide until EDTA is completely dissolved.
  3. Adjust pH back to 4.0 with concentrated hypochloric acid.
  4. Fill up with water to 800 ml and dissolve following components:
    1. 10 g magnesium chloride sexahydrate
    2. 200 mg zinc sulfate heptahydrate
    3. 100 mg calcium chloride dihydrate
    4. 500 mg iron(II) sulfate heptahydrate
    5. 20 mg sodium molybdate dihydrate
    6. 20 mg copper(II) sulfate quintahydrate
    7. 40 mg cobalt(II) chloride sexahydrate
    8. 100 mg manganese(II) chloride sexahydrate

For 1x medium without carbon source, pool 10 ml of each 100x stock solution and fill up to 1000 ml with sterile, deionized water and store at 4°C.

M9 Minimal Medium (M9)

Components for 1 L Volume
bidest. water 778.667 ml
10x Salt solution 100 ml
Magnesiumsulfatehaptahydrate (10 mM) 100 ml
Glucose 20% (w/v) 20 ml
1000x Trace elements 1 ml
Thiamin (1 mM) 0.333 ml


10x Salt solution
Component' Final concentration Concentration in stock solution
BisTris 95 mM 200,000 mg/L
Ammmonium chloride 60 mM 32,100 mg/L
Sodium citrate 12.5 mM 27,000 mg/L
Monopotassium phosphate 3 mM 4,170 mg/L
Dipotassium phosphate 0.7 mM 1,590 mg/L
1000x Trace elements
ComponentFinal concentration Concentration in stock solution
Iron(III) chloride 50 mM 13,515 mg/L
Calcium chloride 20 mM 2,220 mg/L
Manganese(II) chloride 10 mM 1,258 mg/L
Zinc sulfate 10 mM 1,615 mg/L
Cobalt(II) chloride 2 mM 260 mg/L
Copper(II) chloride 2 mM 269 mg/L
Nickel(II) chloride 2 mM 259 mg/L
Sodium molybdate 2 mM 412 mg/L
Sodium selenite 2 mM 346 mg/L
Boric acid 2 mM 124 mg/L
Hydrochloric acid 1 mM 20 ml

Transformation Medium

Super Optimal broth with Catabolite repression medium (SOC)

  1. components
    0,5% yeast extract
    2% tryptone
    10 mM NaCl
    2.5 mM KCl
    20 mM MgSO4
  2. fill up with deionized water
  3. adjust to pH 7.5 with NaOH
  4. after autoclaving, add 20 mM sterile glucose solution (filter sterilization)

References

  • Hartmans, S., Smiths J.P., Volkering F., and de Brondth, J.A. (1989). Metabolism of Styrene Oxide and 2-Phenylethanol in the Styrene-Degrading Xanthobacter Strain 124X. Applied and Environmental Microbiology, Nov. Available at: http://www.ncbi.nlm.nih.gov/pubmed/?term=PMC203180.