OHS026
Safe Work Procedure
Faculty/Division
School/ Divisional Unit
Medicine
POWCS/ORC
Document number
POWCS.ORC.SWPM5
Initial Issue date
18/03/2008
Current version
2
Current Version
Issue date
18/03/2008
Next review date
01/12/2010
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:
Associated risk assessment title and location: POWCS.ORC.RA M5
Describe the activity or process
This SWP describes the correct procedure for making up various broths and 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; SWP 16 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 reagent that requires melting before use (i.e. anything containing agar) should be dispensed
into 500 mL bottles prior to autoclaving; otherwise it will not fit in the microwave (NB: - this constitutes
a burn hazard)
(iii) Any solution filling a bottle to greater than 70% capacity risks boiling over during autoclaving
(iv) For best results, L-broth and LB agar should be made fresh. It is recommended that LB agar not
be kept for longer than 1 month
A. BUFFERS
1. EDTA stock solutions: - ethylene diamine tetraacetic acid, disodium salt (MW = 372.2) is used to
prepare stock solutions for molecular biology techniques; it will not go completely into solution unless the
pH is adjusted to 8.0 with NaOH
(NB: - NaOH is corrosive)
______________________________________________________________________________________________________________________
Page 1 of 5
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
Describe the activity or process
2. Tris-EDTA buffer (TE buffer): - to make up 200 mL
- Tris, pH 8.0, 10 mM (2 mL of 1 M stock)
- EDTA 1 mM
(800 µL of 0.25 M stock)
- add ddH2O to 200 mL
- sterilise by autoclaving (see SWP A16) and store at room temperature
(NB: - Tris is an irritant)
3. Virion lysis buffer (for calculation of virus particles)
- add 0.1% sodium dodecyl sulphate (SDS) to TE buffer
(NB: - SDS is a hazardous substance)
4. Tris-acetate-EDTA buffer (TAE buffer): - to make up 1000 mL of 50x stock
- Tris base (MW = 121.14) 242 g
- dissolve in 500 mL ddH2O
- add 57.1 mL glacial acetic acid
- add 100 mL 0.5 M EDTA
- add ddH2O to 1000 mL; the pH is not adjusted, but should be ~ 8.5
- the solution can be sterilised if desired (see SWP A16); this is recommended for procedures
involving RNA
- for running gels, dilute 40 mL 50x stock in 1960 mL ddH2O; this gives a final concentration of 40
mM Tris-acetate and 1 mM EDTA.
(NB: - glacial acetic acid is flammable and a hazardous substance)
Solution 1 (for preparation of DNA): - to make 200 mL
- glucose 50 mM
(20 mL of 0.5 M stock)
- Tris, pH 8.0, 25 mM (5 mL of 1 M stock)
- EDTA 10 mM
(8 mL of 0.25 M stock)
- add ddH2O to 200 mL
- sterilise by autoclaving (see SWP A16) and store at 4oC
(NB: - Tris is an irritant)
4. Solution 2 (for preparation of DNA): - to make 10 mL (NB: - this must be prepared fresh each time)
- NaOH 0.2 M
(2.0 mL of 1 M stock)
- 1% SDS
(1.0 mL of 10% stock)
- ddH2O
(7.0 mL)
(NB: - NaOH and SDS are hazardous substances)
5. Solution 3 (for preparation of DNA): - to make 100 mL
- potassium acetate
29.44 g
- ddH2O
60 mL
- glacial acetic acid
11.5 mL
- add ddH2O to 100 mL
- store at 4oC
______________________________________________________________________________________________________________________
Page 2 of 5
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
6. QIAGEN buffers (for preparation of DNA) (NB: - these are purchased either as part of a kit or as
individual reagents: P1, cat. # 19051; P2, cat. # 19052; P3, cat. # 19053; QBT, cat. # 19054; QC, cat. #
19055; QF, cat. # 19056; QN is no longer sold separately)
Buffer:
Composition:
Store at:
Buffer P1
(resuspension buffer)
50 mM Tris-Cl, pH 8.0
10 mM EDTA
100 g/mL RNase A
Room temperature, or at
4oC after the addition of
RNase A
Buffer P2
(lysis buffer)
200 mM NaOH
1% SDS (w/v)
Room temperature
Buffer P3
(neutralisation buffer)
3.0 M potassium acetate, pH 5.5
10-25% acetic acid
Room temperature or 4oC
Buffer QBT
(equilibration buffer)
750 mM NaCl
50 mM MOPS, pH7.0
15% v/v isopropanol
0.15% v/v Triton X-100
Room temperature
Buffer QC
(wash buffer)
1.0 M NaCl
50 mM MOPS, pH 7.0
15% v/v isopropanol
Room temperature
Buffer QF
(elution buffer)
1.25 M NaCl
50 mM Tris-Cl, pH 8.5
15% v/v isopropanol
Room temperature
Buffer QN
(alternative elution buffer)
1.6 M NaCl
50 mM MOPS, pH 7.0
15% v/v isopropanol
Room temperature
(NB: - RNase A, buffer P2 and buffer P3 are irritants; buffers QBT, QC, QF and QN are irritants and
flammable)
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 SWP A16) and store at room temperature
2. L-broth agar (LB agar): - to make 1000 mL
 Option A:
- add 15 g Bacto-Agar (cat. # 0140-01) to 1000 mL LB
- sterilise/melt by autoclaving (see SWP A16) and store at room temperature
 Option B:
- add 32 g LB Agar (GE HealthCare/USB cat. # US75851) to 1000 mL ddH2O
- sterilise/melt by autoclaving (see SWP A16) and store at room temperature
3. Terrific broth (T-broth or TB): - to make 1000 mL
- Solution A:
- KH2PO4 0.17 M
2.31 g
- K2HPO4 0.72 M
12.54 g
- dissolve in 90 mL ddH2O; add ddH2O to 100 mL
- sterilise by autoclaving (see SWP A16) and allow to cool
______________________________________________________________________________________________________________________
Page 3 of 5
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
- tryptone
12.0 g
- yeast extract
24.0 g
- glycerol
4.0 mL
- add to 900 mL ddH2O and stir or shake until dissolved
- sterilise by autoclaving (see SWP A16)
- allow to cool to 60oC or less and add 100 mL sterile Solution A
- store at room temperature
4. S.O.C. broth (for transformation of bacteria / preparation of DNA): - to make 1000 mL
(NB: - the terms “S.O.C. medium” and “S.O.C. broth” are often used interchangeably, but S.O.C. medium
does not contain MgSO4. S.O.C. broth can be purchased commercially [Invitrogen, cat. # 15544-034]
under the name “S.O.C. medium”.)
- tryptone
20.0 g
- yeast extract
5.0 g
- NaCl 10 mM
2.0 mL of 5 M stock
- KCl 2.5 mM
2.5 mL of 1 M stock
- MgCl2 10 mM
10 mL of 1 M stock
- MgSO4 10 mM
10 mL of 1 M stock
- add to 900 mL ddH2O and shake or stir until dissolved
- add ddH2O to 1000 mL and sterilise by autoclaving (see SWP A16)
- allow to cool to 60oC or less
- just before use, add 20 mL filter-sterilised 1 M glucose
List all resources required including plant, chemicals,
personal protective clothing and equipment, etc
Latex Gloves, long-sleeve gown, Tris, SDS, NaOH, glacial acetic acid, RNase A and buffers P2 and P3,
QBT, QC, QF and/or QN
List potential hazards and risk controls including specific
precautions required
Hazardous only
Hazardous and corrosive: Sodium Hydroxide, Glacial Acetic acid
List emergency shutdown instructions
List clean up and waste disposal requirements
______________________________________________________________________________________________________________________
Page 4 of 5
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List legislation, standards and codes of practice used in
the development of the SWP
Supervisory approval, training, and review
Supervisor:
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
SWP review date:
Responsibility for SWP review:
______________________________________________________________________________________________________________________
Page 5 of 5
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007