Team:Aachen/Notebook/Protocols/Culture medium and conditions
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<li style="width:106px;margin-left: 12px;margin-right: 12px;" > | <li style="width:106px;margin-left: 12px;margin-right: 12px;" > | ||
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black"> | <a class="menulink" href="https://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 | + | <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(https://static.igem.org/mediawiki/2014/1/10/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(https://static.igem.org/mediawiki/2014/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"> | ||
</div> | </div> | ||
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= Culture Media = | = 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)=== | === Luria-Bertani Medium (LB)=== | ||
# weight components | # weight components | ||
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## '''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 ('''< | + | # for plates, wait until you can touch the bottle ('''<60°C''', clean bench!) |
- | # add antibiotics (1 | + | # add antibiotics (1 µl/ml) and '''shake''' (gloves!) |
- | === | + | === Terrific-Broth-Medium (TB) === |
- | # components 1: | + | # components for 1 : |
- | #: '''4 | + | #: '''4 ml/L glycerol''' |
#: '''12 g/L tryptone''' | #: '''12 g/L tryptone''' | ||
#: '''24 g/L yeast extract''' | #: '''24 g/L yeast extract''' | ||
- | # fill up to 900 | + | # fill up to 900 ml with deionized water |
# '''mix well''' by shaking | # '''mix well''' by shaking | ||
# autoclave | # autoclave | ||
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#: '''0.17 M KH<sub>2</sub>PO<sub>4</sub>''' | #: '''0.17 M KH<sub>2</sub>PO<sub>4</sub>''' | ||
#: '''0.72 M K<sub>2</sub>HPO<sub>4</sub>''' | #: '''0.72 M K<sub>2</sub>HPO<sub>4</sub>''' | ||
- | # dissolve in 100 | + | # dissolve in 100 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 L | ||
+ | #: '''Enzymatic Digest of Gelatin 5 g''' | ||
+ | #: '''Beef Extract 3 g''' | ||
+ | #: '''Agar 15 g''' | ||
+ | # Final pH: 6.8 ± 0.2 at 25°C | ||
+ | # Suspend 23 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 min. | ||
+ | == Minimal Media == | ||
=== Hartmans Minimal Medium (HM) === | === Hartmans Minimal Medium (HM) === | ||
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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 g dipotassium phosphate, | + | * '''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 ammonium sulfate''' composed of 200 g/l ammonium sulfate and sterilized by autoclaving. | ||
* '''100x MM salts''' | * '''100x MM salts''' | ||
# Add 1 g EDTA to 25 ml water. | # Add 1 g EDTA to 25 ml water. | ||
- | # Add drops of 10 M sodium hydroxide until EDTA is completely dissolved. | + | # Add drops of 10 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 ml and dissolve following components: | # Fill up with water to 800 ml and dissolve following components: | ||
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! '''Components for 1 L''' !! '''Volume''' | ! '''Components for 1 L''' !! '''Volume''' | ||
|- | |- | ||
- | | bidest. water || style="text-align:right"| 778.667 | + | | bidest. water || style="text-align:right"| 778.667 ml |
|- | |- | ||
- | | 10x Salt solution || style="text-align:right"| 100 | + | | 10x Salt solution || style="text-align:right"| 100 ml |
|- | |- | ||
- | | Magnesiumsulfatehaptahydrate (10 mM) || style="text-align:right"| 100 | + | | Magnesiumsulfatehaptahydrate (10 mM) || style="text-align:right"| 100 ml |
|- | |- | ||
- | | Glucose 20% (w/v) || style="text-align:right"| 20 | + | | Glucose 20% (w/v) || style="text-align:right"| 20 ml |
|- | |- | ||
- | | 1000x Trace elements || style="text-align:right"| 1 | + | | 1000x Trace elements || style="text-align:right"| 1 ml |
|- | |- | ||
- | | Thiamin (1 mM) || style="text-align:right"| 0.333 | + | | Thiamin (1 mM) || style="text-align:right"| 0.333 ml |
|} | |} | ||
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| Boric acid || style="text-align:right"| 2 mM || style="text-align:right"| 124 mg/L | | Boric acid || style="text-align:right"| 2 mM || style="text-align:right"| 124 mg/L | ||
|- | |- | ||
- | | Hydrochloric acid || style="text-align:right"| 1 mM || style="text-align:right"| 20 | + | | Hydrochloric acid || style="text-align:right"| 1 mM || style="text-align:right"| 20 ml |
|- | |- | ||
|} | |} | ||
</center> | </center> | ||
- | === | + | == Transformation Medium == |
- | + | ||
=== Super Optimal broth with Catabolite repression medium (SOC) === | === Super Optimal broth with Catabolite repression medium (SOC) === | ||
# components | # components | ||
- | #: '''0,5 | + | #: '''0,5% yeast extract''' |
- | #: '''2 | + | #: '''2% tryptone''' |
#: '''10 mM NaCl''' | #: '''10 mM NaCl''' | ||
#: '''2.5 mM KCl''' | #: '''2.5 mM KCl''' | ||
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# after autoclaving, add 20 mM sterile glucose solution (filter sterilization) | # 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. | ||
<html> | <html> | ||
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<li style="width:106px;margin-left: 12px;margin-right: 12px;" > | <li style="width:106px;margin-left: 12px;margin-right: 12px;" > | ||
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black"> | <a class="menulink" href="https://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 | + | <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(https://static.igem.org/mediawiki/2014/1/10/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(https://static.igem.org/mediawiki/2014/1/10/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
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