OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
STGCL.SWP.47.1
School/ Divisional Unit
STGCL
Initial Issue date
25/03/10
Current version
1.0
Current Version
Issue date
25/03/2010
Next review date
March 2012
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: Plasmid extraction using Wizard® Plus SV Minipreps DNA Purification System (Promega)(Miniprep)(Cat. No.:A1460) and with
PureLinkTM HiPure Plasmid DNA Purification Kits (invitrogenTM)(Midi or Maxiprep)(Cat. No.: K2100-07).
Description: This Safe Work Procedure describes the extraction of plasmids using extraction kit from Promega and invitrogenTM
Associated risk assessment title and location: STGCL.RA.47.1
Describe the activity or process
This SWP describes the protocols for isolating plasmid DNA from bacterial cell cultures. These procedures are performed in the PC-2
facility following the OGTR guidelines. PC-2 induction must be carried out before these methods are undertaken.
When producing DNA for restriction enzyme screening, a mini-prep is used. Once the identity and the quality of the plasmid have
been confirmed, larger amounts of DNA for experimental use are generated using either a midi- or a maxi-prep (depending upon
your requirements).
These protocols involve the use of hazardous substances. The attached Risk Assessment and all relevant MSDSs must be
accessed and read before the work is commenced. In addition, this document references STGCL.SWP.46.1: “Broths and buffers for
molecular biology” and STGCL.SWP.49.1: “Preparation of competent bacteria and transformation by heat shock”; be aware that the
hazards associated with the procedures described in those documents will not be detailed here. Furthermore, all persons carrying
out these protocols must also be familiar with STGCL.SWP.2.1, which describe the correct procedures to be followed when disposing
of PC-2 waste.
NB: - in this document, preparation of DNA using commercial kits will be described. Using a kit is quicker, and involves less exposure
to hazardous substances.
These methods are described in the singular, however you should never make just one preparation! Even numbers make
centrifuging simpler.
A1. Mini-prep DNA using Wizard® Plus SV Minipreps DNA Purification System (Promega)
1.
Working in a biohazard hood, transfer 10 mL LB into a 50 mL centrifuge tube, then add the required concentration of antibiotic.
(NB: - the antibiotic that you add and its concentration will be determined by the nature of your plasmid)
2.
Inoculate the LB with a single colony from an agar plate or 10 µL of thawed glycerol stock. Incubate overnight (15-17 hr, no
longer) at 37oC in the shaking incubator at 225 rpm. All cultures must be labeled with the full plasmid name and the type of
bacteria (e.g. p922KAICAT in XL1 Blue E. coli), as well as your name and the date.
3.
Working in a biohazard hood, harvest 1-5ml (high-copy-number plasmid) or 10ml (low-copy-number plasmid) of bacterial
culture by centrifugation for 5 minutes at 10,000 g for in a tabletop centrifuge. Pour off the supernatant.
4.
Decant the supernatant into an appropriate container for decontamination by bleaching.
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Describe the activity or process
5.
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Add 250µl of Cell Resuspension Solution and completely resuspend the cell pellet by vortexing or pipetting. It is essential to
thoroughly resuspend the cells. Transfer the resuspended cells to a sterile 1.5ml microcentrifuge tube(s).
Add 250µl of Cell Lysis Solution and mix by inverting the tube 4 times (do not vortex). Incubate until the cell suspension
clears (approximately 1-5 minutes).
Add 10µL of Alkaline Protease Solution and mix by inverting the tube 4 times. Incubate for 5 minutes at room temperature.
Add 350µL of Neutralization Solution and immediately mix by inverting the tube 4 times (do not vortex).
Centrifuge the bacterial lysate at maximum speed (around 14,000 g) in a microcentrifuge for 10 minutes at room temperature.
Transfer the cleared lysate (approximately 850µl) to the prepared Spin Column by decanting. Avoid disturbing or transferring
any of the white precipitate with the supernatant.
Centrifuge the supernatant at maximum speed in a microcentrifuge for 1 minute at room temperature. Remove the Spin
Column from the tube and discard the flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection
Tube.
Add 750µL of Column Wash Solution, previously diluted with 95% ethanol, to the Spin Column.
Centrifuge at maximum speed in a microcentrifuge for 1 minute at room temperature. Remove the Spin Column from the tube
and discard the flowthrough. Reinsert the Spin Column into the Collection Tube.
Repeat the wash procedure using 250µL of Column Wash Solution.
Centrifuge at maximum speed in a microcentrifuge for 2 minutes at room temperature.
Transfer the Spin Column to a new, sterile 1.0ml microcentrifuge tube.
Elute the plasmid DNA by adding 100µL of Nuclease-Free Water to the Spin Column. Centrifuge at maximum speed for 1
minute at room temperature in a microcentrifuge.
Cap the microcentrifuge tube and store the purified plasmid DNA at -20oC or below.
B1. Midi-prep DNA using PureLinkTM HiPure Plasmid DNA Purification Kits (invitrogenTM)
1.
Working in a biohazard hood, transfer 100 mL LB into a 250 mL conical flask, then add the required concentration of
antibiotic. (NB: - the antibiotic that you add and its concentration will be determined by the nature of your plasmid)
2.
Inoculate the LB with 100 L glycerol stock or 100 L of a pre-screened small-scale culture. Incubate overnight (15-17 hr, no
longer) at 37oC in the shaking incubator at 180 rpm. All cultures must be labeled with the full plasmid name and the type of
bacteria (e.g. p922KAICAT in XL1 Blue E. coli), as well as your name and the date.
3.
Working in a biohazard hood, for high copy number plasmids, use 15-25ml of an overnight LB culture per sample in a
disposable 50-ml disposable tube. For low copy number plasmids, use 25-100ml of an overnight LB culture per sample in a
50-ml disposable tube.
4.
Working in a biohazard hood, harvest the cells by centrifuging the overnight LB-culture at 4,000 g for 10 minutes in a bucket.
Remove all medium.
5.
Add 4ml Resuspension Buffer (R3) with RNaseA to the cell pellet and resuspend the cells until homogeneous. Transfer the
cell suspension to a 15-ml centrifuge tube.
6.
Add 4ml Lysis Buffer (L7). Mix gently by inverting the capped tube five times. Do not vortex. Incubate at room temperature for
5 minutes.
7.
Add 4ml Precipitation Buffer (N3) and mix immediately by inverting the capped tube until the mixture is homogeneous. Do not
vortex.
8.
Centrifuge the mixture at >12,000g for 10 minutes at room temperature.
9.
Load the supernatant onto the equilibrated column. Allow the solution in the column to drain by gravity flow.
10. Wash the column twice with 10ml Wash Buffer (W8). Allow the solution in the column to drain by gravity flow after each wash.
Discard the flow-through.
11. Place a sterile 15-ml centrifuge tube (elution tube) under the column.
12. Add 5ml Elution Buffer (E4) on the column to elute the DNA. Allow the solution to drain by gravity flow. Do not force out any
remaining solution.
13. Add 3.5ml isopropanol to the elution tube. Mix well.
14. Centrifuge the tube at >15,000 g for 30 minutes at 4oC. Carefully remove and discard the supernatant.
15. Resuspend the pellet in 3ml 70% ethanol.
16. Centrifuge the tube at >15,000 g for 5 minutes at 4oC. Carefully remove and discard the supernatant.
17. Air-dry the pellet for 10 minutes.
18. Resuspend the DNA pellet in 200L TE Buffer (TE). For low copy number plasmids, use 100L of TE buffer.
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Describe
the activity or process
B2. Maxi-prep DNA using PureLinkTM HiPure Plasmid DNA Purification Kits (invitrogenTM)
1.
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Working in a biohazard hood, transfer 200 mL LB into a 500 mL conical flask, then add the required concentration of
antibiotic. (NB: - the antibiotic that you add and its concentration will be determined by the nature of your plasmid)
Inoculate the LB with 100 L glycerol stock or 100 L of a pre-screened small-scale culture. Incubate overnight (15-17 hr, no
longer) at 37oC in the shaking incubator at 225 rpm. All cultures must be labeled with the full plasmid name and the type of
bacteria (e.g. p922KAICAT in XL1 Blue E. coli), as well as your name and the date
Working in a biohazard hood, 200 mL culture are transferred into 500 mL centrifuge pots; it is necessary to prepare even
numbers, so that they will balance.
Harvest the cells by centrifuge at 5000 g for 10 min at 4oC; in the hood, decant the supernatant into the empty conical flask
and bleach.
Add 10ml Resuspension Buffer (R3) with RNaseA to the cell pellet and resuspend the cells until homogeneous. Transfer the
cell suspension to a 15-ml centrifuge tube.
Add 10ml Lysis Buffer (L7). Mix gently by inverting the capped tube five times. Do not vortex. Incubate at room temperature
for 5 minutes.
Add 10ml Precipitation Buffer (N3) and mix immediately by inverting the capped tube until the mixture is homogeneous. Do
not vortex.
Centrifuge the mixture at >12,000g for 10 minutes at room temperature.
Load the supernatant onto the equilibrated column. Allow the solution in the column to drain by gravity flow.
Wash the column twice with 60ml Wash Buffer (W8). Allow the solution in the column to drain by gravity flow after each wash.
Discard the flow-through.
Place a sterile 30-ml centrifuge tube (elution tube) under the column.
Add 15ml Elution Buffer (E4) on the column to elute the DNA. Allow the solution to drain by gravity flow. Do not force out any
remaining solution.
Add 10.5ml isopropanol to the elution tube. Mix well.
Centrifuge the tube at >15,000 g for 30 minutes at 4oC. Carefully remove and discard the supernatant.
Resuspend the pellet in 5ml 70% ethanol.
Centrifuge the tube at >15,000 g for 5 minutes at 4oC. Carefully remove and discard the supernatant.
Air-dry the pellet for 10 minutes.
Resuspend the DNA pellet in 500L TE Buffer (TE). For low copy number plasmids, use 200L of TE buffer.
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Date Effective: 01/01/2007
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List all resources required including
plant, chemicals, personal protective
clothing and equipment, etc
Chemicals
 L-broth (LB)
 TE buffer
 Appropriate selection agent for your plasmid
 Agar plate(s) with colonies or a glycerol stock
 95% and 70% v/v ethanol (NB: - this is a hazardous and flammable substance)
 Isopropanol (NB: - this is a hazardous and flammable substance)
 Buffer R3
 Buffer L7
 Buffer N3
 Buffer EQ1
 Buffer W8
 Buffer E4
 Cell Lysis Solution
 Cell Resuspension Solution
 Alkaline Protease
 Neutralization Solution
 Column Wash Solution
Personal Protective Clothing:
 Wear suitable protective clothing (i.e. long-sleeve gown), latex gloves, and eye protection.
Plant:

Class I Biohazard Safety Cabinet

Centrifuge Hermle Z 513 K

Conical Flask

Minispin Eppendorf Centrifuge

Orbital Shaking Incubator
List potential hazards and risk controls
including specific precautions required
Hazards: Addition of antibiotics to Lauria Broth (LB) broth culture where the antibiotics are hazardous substances
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves.
Hazards: Handling of bacteria culture outside of a Class II Biohazard Safety Cabinet risks generating aerosol particles which may be
inhaled or ingested.
Risk Controls: An SWP has been prepared. External and internal PC-2 training is compulsory. Wear suitable protective clothing,
goggles and gloves.
Hazards: Handling of 70% ethanol and 95% ethanol which is highly flammable and irritant to eyes.
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves. Keep away from sources of ignition. Do
not breath vapour. Keep container tightly closed.
Hazards: Handling of isopropanol which is highly flammable, irritant to eyes. Vapours cause drowsiness and dizziness.
Risk Controls: MSDSs are available. Wear suitable protective clothing, goggles and gloves. Keep away from sources of ignition. Do
not breath vapour, mist or gas. Keep container tightly closed.
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.
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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
Isopropanol, ethanol waste has to be collected separately and collected as chemical waste.
All waste must be collected, treated if required 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 Induction

STGCL PC-2 Induction
SWP review date: March 2012
Responsibility for SWP review: Jaa Yien New
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Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007