1 MPACC Fermentation Suite CB12 Lab Management Plan 2019/2020 For members wishing to do recombinant protein expression in E.coli or yeast systems, the fermentation suite contains 6 Infors orbital incubators, a Class II Safety Cabinet, three Beckman Avanti centrifuges, sonicator and a high-pressure homogenizer. Our Prep room Technician, Anita Andreou [email protected]can prepare and autoclave standard media flasks (£5/per flask charge for all consumables) and we also have antibiotic specific agar plates available (£1/per plate). Administrator: Annette Steward [email protected]Any project involving ANY biological organisms or biological samples must be assessed and approved by the Biological Safety committee PRIOR to obtaining samples and beginning work. Please consult https://www.ch.cam.ac.uk/safety/biological-safety and contact the Departmental Biological Safety Officer, Dr. Richard Turner [email protected]Permitted work in the MPACC is only with ACDP Hazard Group 1 organisms. Please note all our laboratories are containment level 1 specifications. Label all culture flasks, bottles of media, plates, tubes with your CRSID and date Unlabelled/unidentified samples will be discarded Any work with Biological organisms (genetically modified and non-geneticaly modified) and biological samples must be inactivated prior to disposal in general waste, by autoclaving or disinfecting with Fam30 or 70% ethanol. Please follow your Biological Risk Assessment instructions You are required to wear a lab coat when working with micro-organisms You are not required to wear gloves when using E. coli, however you must always wash your hands afterwards Surfaces should be disinfected with 70% ethanol before and after use The Class II safety cabinet in the Fermentation lab is for use with micro organisms only e.g. E.coli/yeast – do not use for C. elegans (there is a dedicated cabinet in C. elegans lab CB13) For decontamination of large volumes of biological waste use Fam 30 diluted 1:100 – maximum time required 20 minutes – discard down the sink with excess water
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MPACC Fermentation Suite CB12 Lab Management Plan 2019/2020
For members wishing to do recombinant protein expression in E.coli or yeast systems, the fermentation suite contains 6 Infors orbital incubators, a Class II Safety Cabinet, three Beckman Avanti centrifuges, sonicator and a high-pressure homogenizer. Our Prep room Technician, Anita Andreou [email protected] can prepare and autoclave standard media flasks (£5/per flask charge for all consumables) and we also have antibiotic specific agar plates available (£1/per plate). Administrator: Annette Steward [email protected]
Any project involving ANY biological organisms or biological samples must be assessed and approved by the Biological Safety committee PRIOR to obtaining samples and beginning work. Please consult https://www.ch.cam.ac.uk/safety/biological-safety and contact the Departmental Biological Safety Officer, Dr. Richard Turner [email protected]
Permitted work in the MPACC is only with ACDP Hazard Group 1 organisms. Please note all our laboratories are containment level 1 specifications.
Label all culture flasks, bottles of media, plates, tubes with your CRSID and date
Unlabelled/unidentified samples will be discarded
Any work with Biological organisms (genetically modified and non-geneticaly modified) and biological samples must be inactivated prior to disposal in general waste, by autoclaving or disinfecting with Fam30 or 70% ethanol. Please follow your Biological Risk Assessment instructions
You are required to wear a lab coat when working with micro-organisms
You are not required to wear gloves when using E. coli, however you must always wash your hands afterwards
Surfaces should be disinfected with 70% ethanol before and after use
The Class II safety cabinet in the Fermentation lab is for use with micro organisms only e.g. E.coli/yeast – do not use for C. elegans (there is a dedicated cabinet in C. elegans lab CB13)
For decontamination of large volumes of biological waste use Fam 30 diluted 1:100 – maximum time required 20 minutes – discard down the sink with excess water
Do not use the autoclave for decontamination of large volumes of biological waste, unless you suspect a phage infection in your culture – if this is so, report it immediately to [email protected]
If there is spillage in a centrifuge or rotor, wipe up the liquid with paper towels (discard in the Biological Waste Bin) and then disinfect with 70% Ethanol
Rinse out all decontaminated glassware and centrifuge pots/tubes on same day you used it
Place glassware in washing-up bins in media prep room CB16
Do not store samples in centrifuge bottles/tubes; do not store these in freezers as it affects their integrity
Check how many agar plates there are - if you need a large number then order in specially (ask Anita Andreou [email protected])
The different centrifuge pots/tubes require different cleaning procedures
I L centrifuge pots disinfect and clean with 70% Ethanol – do not put in dishwasher
500 ml centrifuge pots and 50 ml centrifuge tubes can be disinfected with Fam 30 (diluted 1:100) and can be cleaned in dishwasher
Report suspected phage infections to [email protected] immediately. If possible - autoclave infected cultures without opening the flasks
MPACC autoclaves
The large Priorclave autoclave can only be operated by users trained by Anita Andreou ([email protected]) - the number of trained users is limited to 2 per group. A small medical autoclave is available for use by MPACC members. Contact [email protected] for training
Media Anita Andreou [email protected] will make the following media for us:
Agar plates - 2xYT-Amp and 2xYT-Kan (stored in cold room CB14)
1 L flasks - 2xYT and LB
Small 2xYT and LB bottles (stored in cold room CB14) Research groups are charged £1 per plate and £5 per flask, unless you pay for and provide your own media
To request flasks write your name, CRSid, research group, date and number of flasks required in the media request book next to the autoclave If you make up your own flasks using MPACC media, you must also record this in the media request book LB agar plates are stored in MPACC cold room – write your name, CRSid, research group, date and number of plates in the log book Appendix Associated risks, general precautions and safe operating procedures for: Fermentation Suite CB12 Infors shaking incubators Beckmann J-26 XPI centrifuges Sonicator Cold Room CB14 High Pressure Homogenizer
Imbalanced rotor can cause leakage of sample and damage centrifuge
Rotor not correctly secured to spindle can damage centrifuge
Centrifuge rotors are heavy
Inappropriate volumes can result in damage to centrifuge pots and subsequent damage to the centrifuge
General precautions and Standard Operating Procedure Ensure the rotor is placed correctly on the centrifuge spindle
Check that none of the rotor chambers contain any liquid from a previous run or from cleaning JLA-8.1000 and JA-25.50 rotors are heavy so take care when lifting these Check integrity (e.g. cracks) and cleanliness of the rotor, pots, rotor adaptors and O-rings before use Pots/tubes including the lids should be balanced on the scales – place opposite each other in the rotor once balanced It is essential that all pots and tubes are balanced on the scales with their respective lids and rotor adapters
Screw lids on firmly to prevent leakage The 1 L pots need to be filled with over 500 ml of liquid - volumes less than 500 ml can cause the 1 L pots to collapse during centrifugation The 1 L pots have a sealing assembly consisting of a brown lid (some have O-rings) and a plastic insert with O-ring
Use the lid-tightening tool for 1 L pots For volumes less than 500 ml use the 500 ml pots for centrifugation When filling the tubes/pots, decontaminate any spills using 70% ethanol THE ROTOR AND LID MUST BE SECURED ONTO THE SPINDLE BEFORE COMMENCING A RUN – MAKE SURE THE ROTOR LID IS SCREWED ON FIRMLY
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Enter the rotor ID, run time, temperature and required speed. Note the maximum speeds of rotors:
JLA-8.1000 max 8000 rpm JLA-10.500 max 10000rpm JA-25.50 max 25000 rpm
***Don’t forget to fill in the log book*** Wait until the centrifuge has reached the required speed If at any time you wish to stop the run, press the STOP button If a serious imbalance occurs, e.g. due to a balancing error or leakage from a centrifuge pot, the centrifuge should shut down the run and an error message will appear Check rotor and centrifuge chamber carefully after your run for any spillages All spills and leakages of E. coli or buffer in the rotor and centrifuge chamber must be cleaned up immediately and decontaminated using 70% ethanol DO NOT USE ANYTHING OTHER THAN 70% ETHANOL AND WATER ON THE ROTORS The anodizing will be corroded by e.g. Fam30, Virkon, Decon and will reduce the life of the rotor Salts in buffers will also corrode the anodizing All pots and tubes must be free of cell culture, lysate, pellet before being placed in the dishwasher baskets To clean 1 L pots: decontaminate with 70% ethanol, rinse with water and leave to dry, once dry put them away The 1 L pots are NOT cleaned in the dishwasher 500 mL pots and 50 mL tubes can be decontaminated with Fam30 solution, rinsed and then placed in the blue washing up baskets for dishwasher cleaning
Potential hearing damage through high-intensity ultrasound
Safe handling of live cell cultures e.g. E. coli
Biological hazard of generating aerosols General precautions:
Do not use the sonicator without being trained – contact Annette Steward [email protected]
Always ensure the door of the sonicator cabinet is properly closed Never allow the sonicator tip to operate in air Never touch the tip when the sonicator is running Standard Operating Procedure using standard sonicator tip Before switching on the sonicator, check that the tip is clean and not excessively pitted
Put your sample in a glass beaker e.g. 80 ml sample in 100 ml glass beaker
Place the beaker in an ice bucket that fits on the stand in the sonicator cabinet; the stand is height adjustable
Ensure the sonicator probe is submerged into approximately half the volume of liquid
Always ensure the sonicator tip is sufficiently submerged in your sample or this can cause your sample to foam - this may occur when supporting ice melts and the beaker slips down. It is prudent to regularly check beaker height on long processes
DO NOT allow the tip of the sonicator probe to touch the bottom of the beaker
Close the door on the sonicator cabinet – make sure it’s properly closed Power levels and pulse durations may be varied as required
Typical cycle parameters for 80 ml sample in 100 ml beaker – 40 % amplitude; ON for 15 sec; OFF for 45 sec; TOTAL 3 min
Press START to begin pulsing. To terminate a process early, press STOP
When complete, remove sample from cabinet and switch off machine
Clean the sonicator tip and the cabinet with 70% ethanol and wipe clean ***SIGN THE LOGBOOK*** If you encounter problems with the sonciator tip or need assistance changing the tip, contact Annette [email protected] or Kevin Judd [email protected]
High Pressure Homogeniser – Cold room CB14 Administrators: Annette Steward [email protected] & Kevin Judd [email protected] Associated risks:
High pressure system – wear safety glasses at all times
Release of N2 gas – risk of asphyxiation due to oxygen depletion
Safe handling of live cell cultures e.g. E. coli
Prolonged exposure to low temperatures General precautions: This instrument is NOT TO BE USED under any circumstance unless you have received training. Contact Annette Steward ([email protected])
Safety glasses and lab coat must be worn at all times whilst using the instrument
An oxygen level monitor is located within the cold room and is linked to visual (blue beacon) and audio alarms
If at any time these alarms become activated YOU MUST LEAVE THE ROOM immediately
It is recommended to work with the cold room door slightly ajar
Any spills of cell culture or lysate must be cleaned and disinfected immediately with 70% ethanol Before You Start: Before using the instrument ensure you have 1.0 L each of MilliQ water, 70% EtOH and 20% EtOH
Do not use the homogeniser for volumes less than 10ml
If using previously frozen cells please ensure the cells are properly thawed prior to resuspension COMPLETE CELL PELLET RESUSPENSION IS VITAL before you start – any lumps of cells will hinder passage of the sample through the system
If you have any doubts, sonicate your sample for a short period of time beforehand It is important to ensure that no air is allowed to get into the system from the sample chamber - air bubbles will stop the instrument from working
***Don’t forget to fill in the log book*** Standard Operating Procedure It is vital that the pump motor is not started with the system under pressure – the pressure regulator on the small right hand gauge should be in the fully anti-clockwise position before starting (No. 6 in Fig. 1) Open the nitrogen gas supply in the cold room by turning the valve on the wall to the in-line position - the left hand small pressure gauge should reach 100 psi (No. 2 in Fig. 1) Before starting the system, check the sample chamber contains at least 50 ml 20% ethanol Check that the output tube is in a waste collection vessel The pump motor is started by turning the green control valve to the vertical position (No. 4 in Fig. 1) Start the pump motor by running 50 ml 20% ethanol through to almost empty the chamber Flush the system with 50 ml Milli Q water Flush the system with 50 ml sample buffer Take care not to allow air into the system when flushing the system Place your well-suspended cells in the sample chamber Transfer the output tube to your collection vessel and begin to run the cell suspension through the system Set the regulator pressure to approximately 50 psi by turning the small right hand pressure regulator in a clockwise direction (No. 6 in Fig. 1) At 50 psi you should notice a distinct change in the sound – this means the homogenization process is working correctly Note: If there is no distinct change in the sound at 50 psi, homogenization is not taking place – contact Annette Steward [email protected] or Kevin Judd [email protected] To reduce pressure – turn small right hand pressure regulator anti-clockwise (No. 6 in Fig. 1)
Adjust the regulator pressure during the run, if necessary – the pressure required to lyse cells is strain dependant e.g. the large homogenizer pressure dial (No. 14 in Fig. 1) should read ≈ 18K psi for E. coli while for yeast ≈ 25K psi You may wish to run the collected product through the system 2 - 3 times to ensure maximum lysis
On final collection, again return the system to zero pressure by turning the small right hand pressure regulator fully anti-clockwise (No. 6 in Fig. 1) Flush the system through with more buffer to ensure no cells remain Flush the system with 50 ml MilliQ water then 50 ml 70% ethanol and then 50 ml 20% ethanol ensuring that you leave some 20% ethanol in the sample chamber (about a third full is absolutely fine) After flushing, ensure the system has no residual back-pressure:
Close the green valve (horizontal position) that controls the pump motor (No. 4 in Fig. 1)
Close fully the nitrogen flow using the valve on the wall (horizontal position)
Open slightly the green control valve (No. 4 in Fig. 1) to purge the system of any residual nitrogen – the small left hand gauge will go down to zero (No. 2 in Fig. 1).
Close the green valve (horizontal position) At the end of your run: Dispose of the contents of the waste collection flask appropriately (e.g. Fam30 treatment) and disinfect any spills with 70% ethanol Ensure:
The small right hand pressure regulator is in the fully anti-clockwise position (No. 6 in Fig. 1),
The green control valve for the motor pump is in the off (horizontal) position (No. 4 in Fig.1)
The nitrogen is turned off at the wall (valve horizontal position)
