Team:Aachen/Notebook/Protocols/Culture medium and conditions
From 2014.igem.org
(Difference between revisions)
(Created page with "__NOTOC__ {{CSS/Main}} {{Team:Aachen/Stylesheet}} {{Team:Aachen/Header}} <span id="partners"></span> = Molecular biological methods = == Cloning == === Restriction Digest === ==...") |
(→References) |
||
(34 intermediate revisions not shown) | |||
Line 3: | Line 3: | ||
{{Team:Aachen/Stylesheet}} | {{Team:Aachen/Stylesheet}} | ||
{{Team:Aachen/Header}} | {{Team:Aachen/Header}} | ||
- | |||
- | |||
- | |||
- | |||
- | = | + | <html> |
+ | <center> | ||
+ | <ul class="menusmall-grid"> | ||
- | === | + | <li style="width:106px;margin-left: 12px;margin-right: 12px;" > |
+ | <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection" style="color:black"> | ||
+ | <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">2D Detection of<br/>IPTG & HSL</div></div> | ||
+ | <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/22/Aachen_14-10-14_button_chip_manufacturing_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"> | ||
+ | </div> | ||
+ | </a> | ||
+ | </li> | ||
- | + | <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"> | ||
+ | <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> | ||
+ | </a> | ||
+ | </li> | ||
- | + | <li style="width:106px;margin-left: 12px;margin-right: 12px;" > | |
- | + | <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Molecular_biological_methods" style="color:black"> | |
- | + | <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Molecular biological methods</div></div> | |
+ | <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/75/Aachen_14-10-14_Eppi_with_green_cells_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"> | ||
+ | </div> | ||
+ | </a> | ||
+ | </li> | ||
- | < | + | <li style="width:106px;margin-left: 12px;margin-right: 12px;" > |
- | + | <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methods" style="color:black"> | |
- | + | <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Analytical methods</div></div> | |
- | + | <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4d/Aachen_14-10-14_Lense_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"> | |
- | + | </div> | |
- | + | </a> | |
- | + | </li> | |
- | + | ||
- | + | </ul> | |
- | + | ||
- | + | ||
- | + | ||
</center> | </center> | ||
+ | </html> | ||
- | == | + | = 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)=== | ||
+ | # weight components | ||
+ | #: '''5 g/L NaCl''' | ||
+ | #: '''10 g/L tryptone''' | ||
+ | #: '''5 g/L yeast extract''' | ||
+ | #: (15 g/L agar for plates) | ||
+ | # fill up to 1 L with deionized water | ||
+ | # '''mix well''' by shaking | ||
+ | # autoclave | ||
+ | ## autoclaving tape, caps slightly unscrewed | ||
+ | ## base of the pot has to be covered with deionized water | ||
+ | ## close lid | ||
+ | ## heat '''level 3 until the pressure valve opens''' | ||
+ | ## reduce '''heat level to 1.5''' | ||
+ | ## set timer to '''20 minutes''' | ||
+ | ## turn heater off | ||
+ | ## '''wait until the pressure valve retracts''' (30-45 minutes) | ||
+ | ## open, close caps & shake | ||
+ | # for plates, wait until you can touch the bottle ('''<60°C''', clean bench!) | ||
+ | # add antibiotics (1 µl/ml) and '''shake''' (gloves!) | ||
- | === | + | === Terrific-Broth-Medium (TB) === |
- | # | + | # components for 1 : |
- | # | + | #: '''4 ml/L glycerol''' |
- | # | + | #: '''12 g/L tryptone''' |
- | + | #: '''24 g/L yeast extract''' | |
- | + | # fill up to 900 ml with deionized water | |
- | # | + | # '''mix well''' by shaking |
- | # | + | # autoclave |
- | # | + | # components 2: |
+ | #: '''0.17 M KH<sub>2</sub>PO<sub>4</sub>''' | ||
+ | #: '''0.72 M K<sub>2</sub>HPO<sub>4</sub>''' | ||
+ | # dissolve in 100 ml deionized water and sterilize it by passing it through a filter | ||
+ | # 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) === | ||
- | + | 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''' | ||
+ | # Add 1 g EDTA to 25 ml water. | ||
+ | # Add drops of 10 M sodium hydroxide until EDTA is completely dissolved. | ||
+ | # Adjust pH back to 4.0 with concentrated hypochloric acid. | ||
+ | # Fill up with water to 800 ml and dissolve following components: | ||
+ | ## 10 g magnesium chloride sexahydrate | ||
+ | ## 200 mg zinc sulfate heptahydrate | ||
+ | ## 100 mg calcium chloride dihydrate | ||
+ | ## 500 mg iron(II) sulfate heptahydrate | ||
+ | ## 20 mg sodium molybdate dihydrate | ||
+ | ## 20 mg copper(II) sulfate quintahydrate | ||
+ | ## 40 mg cobalt(II) chloride sexahydrate | ||
+ | ## 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) === | ||
<center> | <center> | ||
- | {| class="wikitable" | + | {| class="wikitable centered" |
- | ! | + | ! '''Components for 1 L''' !! '''Volume''' |
|- | |- | ||
- | | | + | | bidest. water || style="text-align:right"| 778.667 ml |
|- | |- | ||
- | | | + | | 10x Salt solution || style="text-align:right"| 100 ml |
+ | |- | ||
+ | | Magnesiumsulfatehaptahydrate (10 mM) || style="text-align:right"| 100 ml | ||
+ | |- | ||
+ | | Glucose 20% (w/v) || style="text-align:right"| 20 ml | ||
+ | |- | ||
+ | | 1000x Trace elements || style="text-align:right"| 1 ml | ||
|- | |- | ||
- | | ''' | + | | Thiamin (1 mM) || style="text-align:right"| 0.333 ml |
+ | |} | ||
+ | |||
+ | |||
+ | {| class="wikitable centered" | ||
+ | | colspan="3"| '''10x Salt solution''' | ||
|- | |- | ||
- | + | ! '''Component''' !!''Final concentration''' !! '''Concentration in stock solution''' | |
|- | |- | ||
- | | | + | | BisTris || style="text-align:right"| 95 mM || style="text-align:right"| 200,000 mg/L |
|- | |- | ||
- | | | + | | Ammmonium chloride || style="text-align:right"| 60 mM || style="text-align:right"| 32,100 mg/L |
+ | |- | ||
+ | | Sodium citrate || style="text-align:right"| 12.5 mM || style="text-align:right"| 27,000 mg/L | ||
+ | |- | ||
+ | | Monopotassium phosphate || style="text-align:right"| 3 mM || style="text-align:right"| 4,170 mg/L | ||
+ | |- | ||
+ | | Dipotassium phosphate || style="text-align:right"| 0.7 mM || style="text-align:right"| 1,590 mg/L | ||
+ | |- | ||
|} | |} | ||
- | |||
- | + | {| class="wikitable centered" | |
- | + | | colspan="3"| '''1000x Trace elements''' | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | {| class="wikitable | + | |
- | + | ||
- | + | ||
- | + | ||
|- | |- | ||
- | + | ! '''Component'''!!'''Final concentration''' !!'''Concentration in stock solution''' | |
- | + | |- | |
- | | style=" | + | | Iron(III) chloride || style="text-align:right"| 50 mM || style="text-align:right"| 13,515 mg/L |
+ | |- | ||
+ | | Calcium chloride || style="text-align:right"| 20 mM || style="text-align:right"| 2,220 mg/L | ||
|- | |- | ||
- | | | + | | Manganese(II) chloride || style="text-align:right"| 10 mM || style="text-align:right"| 1,258 mg/L |
- | | style=" | + | |- |
- | | style=" | + | | Zinc sulfate || style="text-align:right"| 10 mM || style="text-align:right"| 1,615 mg/L |
+ | |- | ||
+ | | Cobalt(II) chloride || style="text-align:right"| 2 mM || style="text-align:right"| 260 mg/L | ||
+ | |- | ||
+ | | Copper(II) chloride || style="text-align:right"| 2 mM || style="text-align:right"| 269 mg/L | ||
|- | |- | ||
- | | | + | | Nickel(II) chloride || style="text-align:right"| 2 mM || style="text-align:right"| 259 mg/L |
- | | style=" | + | |- |
- | | style=" | + | | Sodium molybdate || style="text-align:right"| 2 mM || style="text-align:right"| 412 mg/L |
|- | |- | ||
- | | | + | | Sodium selenite || style="text-align:right"| 2 mM || style="text-align:right"| 346 mg/L |
- | | style=" | + | |
- | | style=" | + | |
|- | |- | ||
- | | | + | | Boric acid || style="text-align:right"| 2 mM || style="text-align:right"| 124 mg/L |
- | | style=" | + | |
- | | style=" | + | |
|- | |- | ||
- | | | + | | Hydrochloric acid || style="text-align:right"| 1 mM || style="text-align:right"| 20 ml |
- | | style=" | + | |
- | | style=" | + | |
|- | |- | ||
|} | |} | ||
</center> | </center> | ||
- | == | + | == Transformation Medium == |
- | + | === Super Optimal broth with Catabolite repression medium (SOC) === | |
- | + | # components | |
+ | #: '''0,5% yeast extract''' | ||
+ | #: '''2% tryptone''' | ||
+ | #: '''10 mM NaCl''' | ||
+ | #: '''2.5 mM KCl''' | ||
+ | #: '''20 mM MgSO<sub>4</sub> ''' | ||
+ | # fill up with deionized water | ||
+ | # adjust to pH 7.5 with NaOH | ||
+ | # 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> | |
+ | <center> | ||
+ | <ul class="menusmall-grid"> | ||
- | == | + | <li style="width:106px;margin-left: 12px;margin-right: 12px;" > |
+ | <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection" style="color:black"> | ||
+ | <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">2D Detection of<br/>IPTG & HSL</div></div> | ||
+ | <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/22/Aachen_14-10-14_button_chip_manufacturing_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"> | ||
+ | </div> | ||
+ | </a> | ||
+ | </li> | ||
- | + | <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"> | ||
+ | <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> | ||
+ | </a> | ||
+ | </li> | ||
- | + | <li style="width:106px;margin-left: 12px;margin-right: 12px;" > | |
- | + | <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Molecular_biological_methods" style="color:black"> | |
+ | <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Molecular biological methods</div></div> | ||
+ | <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/75/Aachen_14-10-14_Eppi_with_green_cells_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"> | ||
+ | </div> | ||
+ | </a> | ||
+ | </li> | ||
- | == | + | <li style="width:106px;margin-left: 12px;margin-right: 12px;" > |
+ | <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methods" style="color:black"> | ||
+ | <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Analytical methods</div></div> | ||
+ | <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4d/Aachen_14-10-14_Lense_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"> | ||
+ | </div> | ||
+ | </a> | ||
+ | </li> | ||
- | + | </ul> | |
+ | </center> | ||
+ | </html> | ||
{{Team:Aachen/Footer}} | {{Team:Aachen/Footer}} |
Latest revision as of 03:42, 18 October 2014
|