OHS026
Safe Work Procedure
Faculty/Division
School/ Divisional Unit
Medicine
STGCL
Document number
Initial Issue date
Current version
STGCL.SWP.46.1
25/03/2010
1.0
Current Version
Issue date
Next review date
March 2012
25/03/2010
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist
in the completion of this form.
Safe Work Procedure Title and basic
description
Title:
Broths and buffers for molecular biology
Description: This Safe Work Procedure decribes the preparation of broths and buffers for the work of molecular biology.
Associated risk assessment title and location: STGCL.RA.46.1
Describe
the activity or process
This SWP describes the correct procedure for making up various broths and composition of buffers that are used in molecular
biology methodology. Due to the nature of the document, the reagents used will not be listed separately. Techniques employing the
solutions described here will be described in other SWPs, and cross-referenced to this document.
A number of the reagents listed are hazardous substances. The attached Risk Assessment and all relevant MSDSs must be
accessed and read before any of these procedures are carried out.
Many of these procedures require the use of the autoclave for sterilisation; STGCL.SWP.14.1 and its associated Risk Assessment
must be accessed and read, and training in the use of the autoclave undergone, before any of these procedures are carried out.
Technical advice:
(i) Buffers or broths should be autoclaved within 2 hr of being prepared, to prevent the possibility of
bacterial contamination/growth within the solution
(ii) Any solution filling a bottle to greater than 70% capacity risks boiling over during autoclaving
(iii) For best results, Lauria Broth (LB) and LB agar should be made fresh. It is recommended that LB agar not be kept for
longer than 1 month
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Page 1 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
Describe
the activity or process
A. Solutions
1 Promega solutions (for preparation of DNA) (NB: - these are purchased as part of a kit; Cat. No.:A1460)
Solutions:
Composition:
Store at:
Cell Lysis Solution (CLA)
0.2M NaOH
1% SDS
Room temperature
Cell Resuspension Solution
(CRA)
50mM Tris-HCl (pH 7.5)
10mM EDTA
100µg/ml RNaseA
Room temperature
Neutralization Solution (NSB)
4.09M guanidine hydrochloride
0.759M potassium acetate
2.12M glacial acetic acid
Room temperature or 4oC
Column Wash Solution (CWA)
Alkaline Protease Solution
Final pH is approximately 4.2
162.8mM potassium acetate
22.6mM Tris-HCl (pH 7.5)
0.109mM EDTA (pH 8.0)
Alkaline Protease
Room temperature
Room temperature
(NB: - RNase A and Cell Lysis Solution (CLA) are irritants; Neutralization Solution (NSB) and Alkaline Protease are harmful and
irritants.)
2. invitrogen buffers (for purification of plasmid DNA) (NB: - these are purchased as part of a kit; Cat. No.: K2100-07)
Buffers:
Composition:
Store at:
Resuspension Buffer (R3)
50mM Tris-HCl, pH8.0
10mM EDTA
20mg/ml of RNaseA in Resuspension Buffer
(R3)
0.2M NaOH
1% (w/v) SDS
Room temperature or at 4oC after
the addition of RNase A
Precipitation Buffer (N3)
3.1M Potassium acetate, pH5.5
Room temperature
Equilibration Buffer (EQ1)
0.1M Sodium acetate, pH5.0
0.6M NaCl
0.15% (v/v) Triton®X-100
Room temperature
Wash Buffer (W8)
0.1M Sodium acetate, pH5.0
825mM NaCl
Room temperature
Elution Buffer (E4)
100mM Tris-HCl, pH8.5
1.25M NaCl
Room temperature
TE Buffer (TE)
10mM Tris-HCl, pH8.0
0.1mM EDTA
Room temperature
Lysis Buffer (L7)
Room temperature
(NB: - RNase A, Precipitation Buffer (N3), Equilibration Buffer (EQ1), Wash Buffer (W8) and Elution Buffer (E4) are irritants)
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Page 2 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
B. BROTHS
1. Luria-Bertani Medium (L-broth or LB-broth or LB): - to make 1000 mL
- NaCl
10.0 g
- tryptone
10.0 g
- yeast extract
5.0 g
- adjust the pH to 7.0
- add ddH2O to 1000 mL
- sterilise by autoclaving (see STGCL.SWP.14.1) and store at room temperature
2. L-broth agar (LB agar): - to make 1000 mL
- add 15 g Agar (Amresco cat. #J637) to 1000 mL LB
- sterilise/melt by autoclaving (see STGCL.SWP.14.1) and store at room temperature
3. S.O.C. medium is purchased commercially [Invitrogen, cat. # 15544-034] under the name “S.O.C. medium”.)
List all resources required including
plant, chemicals, personal protective
clothing and equipment, etc
Chemicals:
 Cell Lysis Solution (CLA)
 Cell Resuspension Solution (CRA)
 Neutralization Solution (NSB)
 Column Wash Solution (CWA)
 Alkaline Protease Solution
 Resuspension Buffer (R3)
 RNaseA
 Lysis Buffer (L7)
 Precipitation Buffer (N3)
 Equilibration Buffer (EQ1)
 Wash Buffer (W8)
 Elution Buffer (E4)
 TE Buffer (TE)
 Luria-Bertani Medium
 L-Broth Agar
 S.O.C. medium
Plant:
 Autoclave Machine
 Conical Flask
 Class I Biohazard Safety Cabinet
 Measuring Cylinder
 Petri Dishes
 Schott Bottle
 Weighing Balance
Personal Protective Clothing:
 Wear suitable protective clothing (i.e. long-sleeve gown), latex gloves, thermo gloves and eye protection.
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Page 3 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List potential hazards and risk controls
including specific precautions required
Hazards: Handling of autoclaved LB agar which may lead to burn hazards while removing the autoclaved LB agar from the autoclave
Risk Controls: Training is provided. Wear suitable protective clothing, goggles and thermo gloves.
Hazards: Handling of autoclaved LB broth may lead to burn hazards while removing the autoclaved LB broth from the autoclave
Risk Controls: Training is provided. Wear suitable protective clothing, goggles and thermo gloves.
Hazards: Handling of Wash Buffer (W8) which are irritant to skin and eyes
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves.
Hazards: Handling of Precipitation Buffer (N3) which are irritant to skin and eyes
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves.
Hazards: Handling of Equilibration Buffer (EQ1) which are irritant to skin and eyes
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves.
Hazards: Handling of Elution Buffer (E4) which are irritant to skin and eyes
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves.
Hazards: Handling of Cell Lysis Solution (CLA) which are irritant to skin and eyes
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves.
Hazards: Handling of Alkaline Protease which are harmful, irritant to skin and respiratory system
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves.
Hazards: Handling of Neutralization Solution (NSB) which are harmful if swallowed and irritant to eyes and skin.
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves.
Wear suitable protective clothing (i.e. long-sleeve gown), latex gloves, and eye protection.
List emergency shutdown instructions
After inhalation: In case of unconsciousness place patient stably in side position for transportation.
After skin contact: Immediately wash with soap, water and rinse thoroughly.
After eye contact: Rinse opened eye for several minutes under running water.
After swallowing: If symptoms persist consult doctor.
List clean up and waste disposal
requirements
All waste must be collected and disposed as chemical waste.
Recommended cleansing agents:80% (v/v) of ethanol, if necessary together with cleansing agents.
List legislation, standards and codes of
practice used in the development of the
SWP
Manual for Promega Wizard Plus SV Minipreps DNA Purification system.
MSDS of Promega Wizard Plus SV Minipreps DNA Purification system.
Manual for PureLinkTM HiPure Plasmid DNA Purification Kits (invitrogenTM)(Midi or Maxiprep).
MSDS of PureLinkTM HiPure Plasmid DNA Purification Kits (invitrogenTM)(Midi or Maxiprep).
Australian/New Zealand Standard, Safety in Laboratories Part 3: Microbiological aspects and containment facilities 2002 (AS/NZS
2243.3:2002)
Supervisory approval, training, and review
Supervisor: Prof. Beng H. Chong
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Plant custodian: Prof. Beng H. Chong
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:

UNSW PC-2 Training

UNSW OGTR Training

STGCL PC-2 Induction
SWP review date: March 2012
Responsibility for SWP review: Jaa Y. New
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Page 4 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007