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| - | =Fermentation=
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| - | ===Quad Parallel Fermentation===
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| - | '''Day 1'''
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| - | SOLUTIONS /APPARATUS
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| - | Acid line use 5% v/v 99.8 sulphuric/water)
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| - | pH probe stored in 3M KCl
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| - | use dilute ammonia (10 %)
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| - | DOT probe must be connected 8 hrs pre-use – typically plug in O/N.
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| - |
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| - | ===Method===
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| - | Prepare 5 L defined media in 5L conical flask or equivalent
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| - | --pH from around 2 up to 6..95 with approx 25% ammonia.
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| - | + acid [to 475 ml RO water, add 25ml 99% sulphuric acid carefully]
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| - | + base [to 300 ml RO water add 200 ml 25% v/v ammonia stock, IN FUME
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| - | HOOD]
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| - | + Filt. Sterile antibiotic stocks
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| - | Autoclave
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| - | 4 x l L conicals with 200 ml defined media in them.
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| - | 4 x 1 L conicals with LB in them. 200 ml each.
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| - | 1 x 2L of defined media into Durans (autoclave) pre-PPG addition
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| - | 3 x Tubing for inoculum transfer.
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| - | 1 x 100 ml Duran of defined media for OD measurement.
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| - | Book shakers.
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| - | Check Oxygen supplies!
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| - | Dry eppendorfs in Oven.
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| - | INOCULATION
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| - | SCHEDULE
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| - | '''Day 2'''
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| - | INOCULATION SCHEDULE
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| - | --use a scrape of glycerol stock to inoculate 5ml complex Luria Bertani broth, no
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| - | antibiotic, grown in 25 ml universal.
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| - |
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| - | --incubate at 30°C in an orbital shaker (200 rpm) overnight.
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| - |
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| - | '''Day 3'''
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| - | INOCULATIONSCHEDULE
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| - | --Use 5 ml LB culture to inoculate 200 ml of LB media, containing relevant
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| - | amount of antibiotic for maintenance of plasmid(s).
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| - | Incubate at 30°C in an orbital shaker (200 rpm) for 4-6 hours to an OD600 1-3.
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| - | Check OD600.
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| - | Weigh eppendorfs! Label fractionation eppendorfs.
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| - | Use 20 ml of over-day LB culture to inoculate 200 ml of defined media,
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| - | containing relevant amount of antibiotic for maintenance of plasmid(s). Incubate
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| - | at 30 C, 18-22 hours. 1.5-2.0
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| - | ''Midday''
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| - | Initialise Fermenter apparatus:
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| - | pH calibrate
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| - | DOT check
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| - |
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| - | -fill fermenter chambers with, typically 900ml media. Add PPG 0.2 ml/L.
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| - |
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| - | --wool/foil all filters etc
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| - |
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| - | --autoclave
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| - |
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| - | prepare/calculate how much antibiotic/inducer is needed.
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| - | End of autoclave run –
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| - | --plug in DOT probes, pH probes
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| - |
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| - | --connect acid / base lines, assess pumps
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| - |
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| - | - connect condenser, etc.
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| - | Connect up laptop, etc.
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| - |
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| - | '''Day 4'''
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| - | Confirm fermenter status – data logging etc, cascade control etc.
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| - | Measure OD600 of starter cultures.
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| - | INOCULATION /FERM.SCHEDULE
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| - | Use autoclaved tubing to suck out required volume of inoculum for fermentation
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| - |
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| - | Acid line use 5% v/v 99.8 sulphuric/water)
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| - |
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| - | pH probe stored in 3M KCl
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| - |
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| - | use dilute ammonia (10 %)
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| - |
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| - | DOT probe must be connected 8 hrs pre-use – typically plug in O/N.
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| - |
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| - | 5 L of defined media
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| - |
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| - | ''ADJUST pH BEFORE AUTOCLAVING!!!''
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| - | Chemical Mass Done?
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| - | (NH4)2SO4 25 g
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| - | NaH2PO4.H2O 14g
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| - | KCl 19.38g
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| - | MgSO4.7H2O 5g
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| - | Trace elements 50ml
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| - | Citric acid. H2O 20g
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| - | Glycerol 150ml OR 450ml
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| - |
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| - | ''ADJUST pH BEFORE AUTOCLAVING!!!''
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