UCL-Fermentation

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(New page: =Fermentation= ===Quad Parallel Fermentation=== '''Day 1''' SOLUTIONS /APPARATUS Acid line use 5% v/v 99.8 sulphuric/water) pH probe stored in 3M KCl use dilute ammonia (...)
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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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===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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--incubate at 30°C in an orbital shaker (200 rpm) overnight.
 
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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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-fill fermenter chambers with, typically 900ml media. Add PPG 0.2 ml/L.
 
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--wool/foil all filters etc
 
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--autoclave
 
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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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--connect acid / base lines, assess pumps
 
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- connect condenser, etc.
 
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Connect up laptop, etc.
 
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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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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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5 L of defined media
 
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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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''ADJUST pH BEFORE AUTOCLAVING!!!''
 

Latest revision as of 20:19, 14 October 2010