OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS.CGM.SWP046
Initial Issue date
30/06/09
School/ Divisional Unit
School of Medical Science, Neuromuscular and
Regenerative Medicine Research Unit
Current version
Current Version
Next review date
1.0
30/06/09
Issue date
30/06/09
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:
Quantitative PCR using the Stratagene Mx3000P
Description: Setting up real-time PCR reactions on the Mx3000P machine
Associated risk assessment title and location: 46_RA_QPCR_SP
Describe the activity or process
Note: This SWP does not include instructions on DNA extraction, preparation of DNA
standards or the synthesis of single stranded cDNA from RNA templates. See associated
SWPs for these elements.
Note: This SWP does not give detailed descriptions of experimental design or instructions on
data input, statistical analysis and result interpretation. These issues require careful
consideration and training before attempting this technique. See the Stratagene website for
support documents and seek training from experienced users.
Preparing the Reactions
Thaw an aliquot of the Brilliant II SYBR Green QPCR master mix
Store it on ice while setting up the reactions.
Following initial thawing of the master mix, store the unused portion at 4°C. Multiple freeze-thaw cycles
should be avoided.
SYBR Green I dye (present in the master mix) is light-sensitive, solutions containing the master mix
should be protected from light whenever possible.
It is prudent to set up a no-template control reaction to screen for contamination of reagents or false
amplification.
There is no need to add reference dye to the reactions. Leave this tube unused.
2. Prepare the experimental reactions by adding the following components:
Experimental Reaction
Nuclease-free PCR-grade water to allow the final volume to reach 25 μl (including experimental
DNA) – Do not add template at this stage
12.5 μl of 2× master mix
x μl of upstream primer (50–150 nM final concentration is recommended)
x μl of downstream primer (50–150 nM final concentration is recommended)
Template DNA should be added in a volume that can be accurately pippetted i.e. 2-5ul
Note: smaller total reaction volumes may also be used by proportional reduction of master mix
components. Verify quality of results empirically.
___________________________________________________________________________________________________________
___________
Page 1 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
3. Gently mix the reaction mix without creating bubbles (do not vortex). Dispense into plate wells
4. Add x μl of experimental template gDNA, cDNA, or plasmid DNA to each experimental reaction.
PCR Cycling Programs
7. Place the reactions in the instrument and run the appropriate PCR program below. This amplification
protocol is recommended initially, but optimization may be necessary for some primer/template systems.
Note For short targets (<300 bp), a 2-step PCR protocol may be considered.
PCR Program for Amplification of Short Targets (50–400 bp)
Cycles
1
Duration of cycle
10 minutes
Temperature
95°C
40
30 seconds
1.0 minute a
95°C
55–60°Cb
1.0 minutesa
72°C
Set the temperature cycler to detect and report fluorescence during both the annealing step and the extension step
of each cycle.
b Choose an appropriate annealing temperature for the primer set used.
a
PCR Program for Amplification of Long Targets (400–900 bp)
Cycles
1
Duration of cycle
10 minutes
Temperature
95°C
40
30 seconds
1.0 minute a
95°C
55–60°Cb
1.5 minutesa
72°C
3.0 minutes
72°C
1
Set the temperature cycler to detect and report fluorescence during both the annealing step and the extension step of
each cycle.
b Choose an appropriate annealing temperature for the primer set used.
a
Performing Real-Time PCR Detection
1) Check to verify that the power status indicator (the lower LED on the front of the instrument) is lit and the
Ready status indicator (the upper LED) is continuously lit (glowing green). A blinking Ready status
indicator indicates an experiment is already in process; if the Ready status indicator is off, the instrument is
not available or ready to run an experiment.
2) Ensure the reaction mix in each well of your reaction plate is free of any bubbles and positioned at the
bottom of the well. If not, centrifuge the plate at ~1000 g for 45 seconds.
3) Open the door located on the front of the instrument by sliding it all the way to the top. To expose the
thermal block, pull forward on the hot-top handle and lift the hot-top up and away from the thermal block.
Place your plate in the plate holder with the last row (row H) facing front. Well A1 should be positioned at
the top-left corner of the thermal block. Make sure the plate is properly aligned in the holder. Close the
hot-top assembly by pressing down the hot-top and pushing the handle back into its original place. Slide
down the door to close.
4) Open the Stratagene MxPro QPCR Software. Click cancel when the New Experiment Options dialog box
appears.
5) Select File � Open. Load the RT2Profiler™ PCR Array Template file. This will load the previously saved
setup to the new plate document. Save the new document under a new filename.
6) Click Start Run to begin the PCR run. Wait for about 30 seconds for the initial priming. The estimated run
time will then appear on the screen.
___________________________________________________________________________________________________________
___________
Page 2 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Equipment
 Mx3000P Instrument and computer
 Microcentrifuge
 96 well PCR reaction plate
Reagents

2X Brilliant II SYBR Green QPCR master mix

Oligonucleotides Forward and Reverse (10uM)

Nuclease-free water

Template DNA (genomic, cDNA or recombinant)
List potential hazards and risk controls including
specific precautions required
1.Stratagene Mx3000P machine and computer
Electrical appliance – electrocution hazard
2.Refrigerated microcentrifuge
Electrical appliance – electrocution hazard
Spinning rotor- physical injury hazard
List emergency shutdown instructions
For electrical appliances, the emergency cutoff switch (red button) is located on the wall
near the entrance to the lab.
List clean up and waste disposal requirements
All solution s should be disposed of in chemical waste containers.
Contaminated solid waste should be disposed of through the biological and chemical waste stream.
List legislation, standards and codes of practice used
in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3-2002. Safety in laboratories. Part 3: Microbiological aspects and containment facilities.
AS/NZS 2243.4: 1998 Safety in Laboratories Part 4; Ionising Radiations
AS/NZS 2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
AS/NZS 2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection
UNSW Hazardous Waste Disposal Procedure
Supervisory approval, training, and review
Supervisor: Edna Hardeman
Signature:
Plant custodian: S.Palmer
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per Training Needs Analysis, Induction to lab including Mx3000P induction training, Training in this SWP.
___________________________________________________________________________________________________________
___________
Page 3 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
___________________________________________________________________________________________________________
___________
Page 4 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007