Preparation of Bulk Plasmid from Bacteria (Maxiprep)

LB broth

Antibiotics usually ampicillin at 100mg/ml Hazardous Substances Policy - Assessment CHEMICAL HAZARD AND RISK ASSESSMENT School of Biosciences

Name of Dr S. Minchin Assessment Number* 1394 supervisor Signature Assessor Rita Godfrey Date of Assessment 4 / 6 / 01 Signature

Notes A School COSHH form in Word is available on the School Server. Available from the Health and Safety Unit. Guidance on making an assessment is given in Making a Chemical Hazard and Risk Assessment. Guidance is also available from Guidance on Completing the Chemical Hazard and Risk Assessment Form. Use a continuation sheet to expand any section of this form in hard copy version.

1 LOCATION OF THE WORK ACTIVITY Lab G10

2 PERSONS WHO MAY BE AT RISK List names where possible All occupants of the lab (Laura Tate, Jo Field, Dong Ling Zeng, Rita Godfrey)

3 ACTIVITY ASSESSED Preparation of bulk plasmid from bacteria (Maxiprep)

4 MATERIALS INVOLVED Attach copies of data sheet(s) NAME AMOUNT HAZARD RISK PHRASES HAZDAT NO*** BIOSCIENCESNO*** max/stored LB broth 2 litres NSH

Antibiotics usually 5ml HARMFUL R42/43 May cause ampicillin at 100mg/ml sensitisation by inhalation and skin contact

Chloramphenicol 10g TOXIC R45 May cause cancer R46 May cause heritable genetic damage R63 Possible risk of harm to the unborn child R42/43 May cause sensitisation by inhalation and skin contact.

Bacterial strain containing 400ul Hazard group plasmid glycerol 1 bacteria and stock plasmid. GMAG approved

TES buffer (30mM Tris, 100ml NSH 5mM EDTA, 50mM NaCl)

250mM EDTA 100ml NSH

Lysozyme 10g No R numbers available but avoid skin contact and inhalation as prolonged or repeated exposure may cause allergic reactions in certain sensitive individuals 10% SDS 100ml IRRITANT R36/38 Irritating to eyes 966 and skin HARMFUL R22 Harmful if swallowed

5M NaCl 100ml NSH

Isopropanol 2.5 litres HIGHLY R11 Highly flammable 127 FLAMMABLE IRRITANT R36 Irritating to the eyes R67 Vapours may cause drowsiness and dizziness

Ethanol 2.5 litres HIGHLY R11 Highly flammable 84 FLAMMABLE

70% Ethanol 50ml HIGHLY R11 Highly flammable 84 FLAMMABLE

Caesium chloride 50g NSH

TE buffer 100ml NSH

Ethidium Bromide 10ml TOXIC R40/21/22 Harmful: 1509 10mg/ml possible risk of irreversibl;e effects in contact with skin and if swallowed R26 Very toxic by inhalation R46 May cause heritable genetic damage n-Butan-1-ol (water 200ml FLAMMABLE R10 Flammable 27 saturated) HARMFUL R20 Harmful by inhalation R37/38 Irritating to respiratory system and skin R41 Risk of serious damage to eyes R67 Vapours may cause drowsiness and dizziness

3M Sodium Acetate 2ml NSH

Phenol/Chloroform/Iso- 200ml TOXIC R24/25 Toxic in contact 176 and amyl alcohol with skin and if swallowed 44 25:24:1 Saturated in R34 Causes burns 100mM Tris pH 8.0 Also CORROSIVE R40 Possible risk of contains 0.1% 8- HARMFUL irreversible effects hyroxyquinoline Category 3 R48/20/22 Harmful: danger Carcinogen of serious damage to health by prolonged exposure through inhalation and if swallowed

5 INTENDED USE** Give brief details and attach protocol/instructions See attached sheet

6 RISKS to HEALTH and SAFETY from INTENDED USE From personal exposure or hazardous reactions. Refer to OELs, flash points, etc., as appropriate. Are pregnant women, breast-feeding mothers especially at risk? There is a risk of contact with chloramphenicol, SDS, ethidium bromide, and phenol/chloroform There is a fire risk from the ethanol, isopropanol and n-butan-1-ol Flash points are Ethanol:13’C; Isopropanol 12’C; n-butan-1-ol:29’C There is also a risk of inhaling fumes from isopropanol, phenol/chloroform and butanol. There is a risk of cuts or ouncture wounds when using needles and razor blades.

7 CONCLUSIONS ABOUT RISKS Is level of risk acceptable? Can risk be prevented or reduced by change of substance/procedure? Are control measures necessary? There are a number of serious risks in this assessment for which control measures are necessary.

8 CONTROL MEASURES Additional to Good Chemical Practice All sources of ignition should be shut off when using ethanol, isopropanol, or butanol. All bottle tops and tube caps etc should be replaced as soon as possible when using phenol/chloroform, isopropanol, butanol and ethidium bromide. Apart from ethidium bromide the aforementioned solvents should not be used in a confined space. There should be adequate ventilation to ensure there is not a build up of fumes. Great care should be taken when weighing out chloramphenicol and lysozyme so as not to generate a dust. Gloves must be worn during this procedure as well. Gloves and protective clothing must be worn when handling ethidium bromide at all times. Protective clothing, gloves and goggles or face mask should be worn when handling phenol/chloroform and disposal into waste bottle should be done in the fume hood to reduce the exposure to the fumes. Polyethylene glycol 300 should be on hand in case of emergency. See assessment number 1360 for more detail on using phenol/chloroform. Great care must be taken when using the needles and razor blades. Keep the protective top on the needles whenever possible and try and keep fingers away from the blade. Some effort is required to cut the top of the Quick-seal centrifuge tube, scoring the tube several times will help to position the blade during cutting. Make sure the blade is sharp as a blunt blade is more likely to result in it slipping and cutting fingers.

9 INSTRUCTION/TRAINING Specify course(s) and/or special arrangements.

10 MONITORING Performance of control measures, Check integrity of gloves before use and that fume hood is working properly.

Personal exposure Health Surveillance

11 WASTE DISPOSAL PROCEDURE See School Server for Approved Procedure Document on specific Chemical Waste Disposal. Waste ethanol, isopropanol and butanol should be put in the Non-Halogenated waste solvent bottle which is then stored in the lab solvent bin until full and then taken down to the Stores for specialist disposal. Waste phenol/chloroform is put in the Halogenated waste solvent bottle. This should be done in the fume hood to minimise the exposure to toxic fumes. The supernatant from spinning the cells should be treated with Presept overnight before pouring down the sink. The concentration of Presept used is 3.5 x 5g tablets per 10 litres. The flasks and tubes used to contain any of the culture should also be soaked overnight before washing up. Any other solutions used here can be poured down the sink with plenty of water. Any tubes used for the phenol/chloroform extraction should be rinsed out with plenty of water whilst wearing protective clothing and goggles or a face mask. The tubes should be held well down in the sink and avoid splashes. After the plasmid band has been removed from the tube after the CsCl gradient run, the top of the tube should be VERY CAREFULLY cut open so that the contents can be removed into the waste Non-Halogenated solvent bottle. Protective clothing, gloves, and goggles or a face shield should be worn during this operation. The empty tubes can then rinsed out with plenty of water under the tap, again avoid splashing. The tubes should then be bagged along with any tissues, syringes (minus needles) etc that are contaminated with ethidium bromide, sealed and placed in the lab bin. The used needles should be placed in the lab sharps bin.

12 REVIEW Enter the date or circumstances for review of assessment (maximum review interval 5 years) June 2006

13 EMERGENCY ACTION TO CONTROL HAZARDS To stabilize situation eg spread absorbant on liquid spill; eliminate sources of ignition, etc. Switch off all sources of ignition if ethanol, isopropanol or butanol is spilled. Small spills can be contained with paper towels but if it is large then the spill kit should be used. Spills of bacterial culture should be disinfected with Presept granules. Phenol/chloroform should be contained using the spill kit or paper towels if it is small. Spillage of the powder of chloramphenicol or lysozyme should be carefully collected up into a bag but avoid generating a dust. Ethidium bromide should be contained using paper towels. This spill is unlikely to be big enough to need the spill kit. The paper towels should then be sealed in a bag before disposal.

TO PROTECT PERSONNEL Evacuation, protection for personnel involved in clean-up, Special First Aid Protective clothing, gloves and goggles or a facemask should be worn during all the clean up procedures mentioned here. For spillages of phenol/chloroform and any of the other solvents mentioned, then non-essential personnel should leave the area which should be well ventilated until all fumes have gone. PEG 300 should be on hand in case of phenol/chloroform spills on the skin. The area affected should be swabbed for 30mins. Large areas will require medical attention. See assessment number 1360

TO RENDER SITE OF EMERGENCY SAFE Clean-up/decontamination Any contaminated spill absorbent is scooped up into a labelled container and the School Safety Officer contacted regarding its disposal. The site of the spillages should be washed with water and any paper towels used to dry the area should be sealed in a bag before placing in the lab bin.

CONTACT Dr S. Minchin PHONE 414 5438

10.10.00 * Prefix T is used for Teaching Assessment Number.

** Please include amount of chemicals used and how.

*** Hazdat No is the UNICOSHH datasheet report number.

Biosciences No is the Biosciences data sheet number.

UNICOSHH IS A CHEMICAL DATABASE ON THE HEALTH AND SAFETY UNIT SERVER. BIOSCIENCES DATA SHEETS ARE AVAILABLE IN THE SCHOOL SAFETY OFFICE.