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 KH2PO4
- 0.72 M K2HPO4
- 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)
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
|
Component | Final 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)
- components
- 0,5% yeast extract
- 2% tryptone
- 10 mM NaCl
- 2.5 mM KCl
- 20 mM MgSO4
- fill up with deionized water
- adjust to pH 7.5 with NaOH
- after autoclaving, add 20 mM sterile glucose solution (filter sterilization)
|