OHS026
Safe Work Procedure
Faculty/Division
School/ Divisional Unit
Medicine
POWCS/ORC
Document number
POWCS.ORC.SWPM6
Initial Issue date
16/06/2006
Current version
1
Current Version
Issue date
Next review date
01/12/2010
18/03/2008
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:
Isolation of plasmid DNA (replaces M5.2, M6.2 and M24.1)
Description:
Associated risk assessment title and location: POWCS.ORC.RA M6
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 SWP M4: “Broths and buffers for molecular biology” and SWP M10: “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 SWP P4 and P5, which describe the correct procedures to be followed
when disposing of PC-2 waste.
NB: - in this document, preparation of DNA both “by hand” and using commercial kits will be described.
Using a kit is quicker, and involves less exposure to hazardous substances, but preparing DNA by hand
gives a much better yield (in terms of both quantity and quality) and is less expensive.
These methods are described in the singular, however you should never make just one preparation! Even
numbers make centrifuging simpler.
A1. Mini-prep DNA
1. Working in a biohazard hood, transfer 5 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 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. Cool the culture on ice; also pre-cool the bench-top centrifuge to 4oC
4. Working in a biohazard hood, fill a labeled 1.5 mL tube near to the top with culture
5. Centrifuge at 12000 g for 10 sec at 4 oC
6. Decant the supernatant into an appropriate container for decontamination by bleaching (see SWP P5)
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Describe the activity or process
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Repeat steps 4, 5 and 6; the rest of the culture can be stored at 4oC for up to 1 week
Still in the hood, add 100 µL Solution 1 containing 5 mg/mL lysozyme. Resuspend the pellet and
incubate on ice for 5 min. While the mixture is incubating, prepare fresh Solution 2.
Working on the bench, add 200 µL Solution 2, mix, and incubate on ice for 10 min
Add 150 µL Solution 3, then centrifuge at 12000 g for 10 min at 4oC
Transfer the supernatant into a new 1.5 mL tube (~400 µL)
Add 200 µL phenol and 200 µL chloroform. Vortex thoroughly and centrifuge at 12000 g for 3 min at
4 oC
Remove the supernatant to a new 1.5 mL tube and add 2 volumes of absolute ethanol
Mix and centrifuge at 20817 g for 10 min at 4 oC
Remove the supernatant, taking care not to disturb the pellet, then use a Thirsty-Stix, cotton bud or
tissue to remove as much residual ethanol as possible
Resuspend the pellet in 100 µL TE buffer
Add 1 µL 10 mg/mL RNaseA and incubate at 37oC for 15 min
Add 40 µL Solution 3 and 260 µL ddH2O. Mix well.
Add 400 µL phenol, vortex thoroughly, and centrifuge at 12000 g for 3 min at 4 oC
Carefully remove the top layer to a new 1.5 mL tube and add 200 µL phenol and 200 µL chloroform.
Vortex thoroughly to mix, and centrifuge at 12000 g for 3 min at 4oC
Carefully remove the top layer to a new 1.5 mL tube and add 400 µL chloroform. Vortex thoroughly
to mix, and centrifuge at 12000 g for 3 min at 4oC
Carefully remove top layer to a new 1.5 mL tube and add 1/10th the existing volume of 3 M
sodium acetate. Mix well.
Add twice the new total volume of absolute ethanol. Centrifuge at 12000 g for 20 min at 4oC
Remove the ethanol, taking care not to disturb the pellet, and gently wash the pellet with 500 µL
70% v/v ethanol. Centrifuge at 12000 g for 2 min at 4oC
Carefully remove the ethanol wash, taking care not to disturb the pellet, then use a Thirsty-Stix,
cotton bud or tissue to remove as much residual ethanol as possible.
Resuspend the DNA pellet in 20 µL TE buffer, warming gently if necessary to help dissolve the
pellet. This DNA can now be used for sequencing or restriction enzyme digests or stored at -20oC
A2. Mini-prep DNA using the QIAGEN alkaline lysis methodology
1. Working in a biohazard hood, transfer 2 mL LB into a 14 mL round-bottom tube and 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 1-10 µL of thawed glycerol stock.
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. pShuttle.CMV.PNP in E.
coli DH10B-alpha), as well as your name and the date
3. Working in a biohazard hood, transfer 1.5 mL of culture into a labeled 1.5 mL tube; the rest of the
culture can be stored at 4oC for up to 1 week
4. Centrifuge at 12200 g for 5 min at room temperature
5. Remove the supernatant, leaving the bacterial pellet as dry as possible. (NB: - it is important to
remove all traces of medium from the bacterial pellet, otherwise the plasmid DNA may be resistant
to cleavage by restriction enzymes)
6. Add 200 L of chilled P1 buffer and resuspend the bacterial pellet by pipetting it up and down
7. Add 200 L of P2 buffer and invert a few times. Incubate at room temperature for 4 min, no longer
than 5 min; timing is crucial. The bacterial lysate will become viscous as large complexes
containing bacterial proteins, broken cell walls, and denatured chromosomal DNA are precipitated
from solution when sodium ions are replaced by potassium ions.
8. Add 200 L of chilled P3 buffer, mix by inversion and incubate on ice for 15 min
9. Centrifuge the lysate at 15000 g for 20 min at 4C
10. Transfer the supernatant into a new labeled 1.5 mL tube and discard the pellet
11. Add 1 mL of absolute ethanol to the tube and mix by inversion. Allow the mixture to stand for 2 min
at room temperature
12. Centrifuge at 15000 g for 15 min at room temperature
13. Slowly and carefully remove the supernatant; do not disturb the pellet
14. Add 600 L of chilled 70% v/v ethanol, then centrifuge at 15000 g at room temperature for 5 min
15. Carefully remove the ethanol wash, taking care not to disturb the pellet, then use a Thirsty-Stix,
cotton bud or tissue to remove as much residual ethanol as possible
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Describe the activity or process
16. Air dry the pellet in a 57C oven (a hybidisation chamber can be used for this purpose), checking for
the presence of any ethanol every minute (1-2 minutes of drying is usually sufficient). Do not overdry the pellet as dehydrated DNA is difficult to dissolve, and it may denature
17. Dissolve the pellet in 30-50 L of sterile ddH2O; it should contain 2-3 (up to 5) g of DNA
18. Store at -20C until ready for use
B1. Midi-prep DNA
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. Cool the culture on ice; also pre-cool the centrifuge to 4oC and label the appropriate number of 250
mL centrifuge pots
4. Working in a biohazard hood, 100 mL culture are transferred into 250 mL centrifuge pots; it is
necessary to prepare even numbers, so that they will balance
5. Centrifuge at 3000 g for 15 min at 4oC; in the hood, decant the supernatant into the empty conical
flask and bleach (see SWP P5)
6. In the hood, add 4 mL Solution 1 containing 5 mg/mL lysozyme. Resuspend the pellet and incubate
on ice for 10 min. While the mixture is incubating, prepare fresh Solution 2.
7. Working on the bench, add 6 mL Solution 2, mix, and incubate on ice for 10 min
8. Add 8 mL Solution 3, mix, and incubate on ice for 10 min; centrifuge at 3000 g for 15 min at 4oC
9. Pack a small funnel with glass wool and decant the supernatant through it into a 50 mL tube; discard
the glass wool as cytotoxic waste
10. Add 17 mL isopropanol, mix, and place at -80oC for 15 min; centrifuge at 3000 g for 15 min at 4oC
11. Discard the supernatant and resuspend the pellet in 2 mL TE buffer; add 2.5 mL 4.4 M lithium
chloride, mix, and incubate on ice for a minimum of 1 hr; centrifuge at 3000 g for 15 min at 4oC
12. Transfer the supernatant to a new 50 mL tube and add 10 mL absolute ethanol; centrifuge at 3000 g
for 15 min at 4oC
13. Remove the ethanol, taking care not to disturb the pellet, and gently rinse the pellet with 70% v/v
ethanol; if the pellet dislodges, centrifuge at 3000 g for 15 min at 4oC; otherwise, slowly and carefully
remove the ethanol wash, then use a Thirsty-Stix, cotton bud or tissue to remove as much residual
ethanol as possible
14. Resuspend the pellet in 400 L TE buffer, transfer to a new 1.5 mL tube, add 10 L 10 mg/mL
RNase A and incubate at 37oC for 15 min
15. Add 20 L 10% SDS, heat at 70oC in a water bath for 10 min, then cool on ice
16. Add 430 L phenol and vortex thoroughly to mix; centrifuge at 12000 g for 3 min at 4 oC
17. Remove the top layer to a new 1.5 mL tube and repeat the phenol extraction; centrifuge at 12000 g
for 3 min at 4oC
18. Remove the top layer to new 1.5 mL tube and add 200 L phenol and 200 L chloroform. Vortex
thoroughly, then centrifuge at 12000 g for 3 min at 4oC
19. Remove the top layer to a new 1.5 mL tube and add 400 L chloroform. Vortex thoroughly, then
centrifuge at 12000 g for 3 min at 4oC
20. Remove the top layer to a new 1.5 mL tube and add 1/10th the existing volume of 3 M sodium
acetate. Mix well.
21. Add twice the new total volume of absolute ethanol; centrifuge at 12000 g for 3 min at 4oC
22. Carefully remove the ethanol and gently wash the pellet with 500 L 70% v/v ethanol; centrifuge at
12000 g for 2 min at 4oC
23. Slowly and carefully remove the ethanol wash, then use a Thirsty-Stix, cotton bud or tissue to
remove as much residual ethanol as possible
24. Resuspend the DNA pellet in 200 L TE buffer. If necessary, warm gently to help dissolve the pellet.
The DNA can now be used for sequencing or restriction enzyme digests, or stored at -20oC
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B2. Midi-prep and Maxi-Prep DNA using the QIAGEN Plasmid Kits
(NB: - the QIAGEN Midi- and Maxi-Prep methods follow essentially the same methodology, except for
differences of scale. In the following protocol, the Midi-Prep procedure will be given in the text, while the
Maxi-Prep procedure will be given in square brackets and bolded – e.g. [Maxi-Prep] )
1. Midi- and Maxi-preps are carried out using bacteria (colonies on plates or cultures stored at 4 oC) that
have already been screened by restriction enzyme digest. A culture can be expanded prior to
commencing the full methodology, by inoculating a single colony or 1 mL culture into 5 mL Terrific
broth (TB) plus the appropriate antibiotic in a 50 mL tube, and incubating at 37oC and 180 rpm for 6-8
hr. Alternatively, 1 mL of a pre-existing culture may be used without pre-expansion. For a Maxi-prep,
it is preferable always to expand first.
2. In a biohazard hood, inoculate 1 mL culture into 50-70 mL [120-150 mL] TB (plus antibiotic) in a 500
mL [1000 mL] conical flask and incubate overnight (15-17 hr, no longer) at 37oC and 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 the hood, prepare a glycerol stock of cultured bacteria by mixing 812.5 L of bacteria from
the flask with 887.5 L of 80% glycerol; store at -20C (NB: - this glycerol stock is merely a “back-up”
in case of later problems; for preparation of stocks for long-term-storage, see SWP M10)
4. Harvest the remainder of the bacterial cells by transferring the culture into 250 mL pots and
centrifuging at 3000 g for 1 hr at 4C
5. Decant the supernatant and bleach (see SWP P5). Invert the open centrifuge bottle to allow all the
supernatant to drain away onto absorbent paper; dispose of this as GMO waste (see SWP P4). The
pellet should be as dry as possible; residual medium can interfere with restriction enzyme cleavage of
the DNA
6. Thoroughly resuspend the pellet in 6 mL [12 mL] buffer P1 containing 100 g/mL RNase A; there
should be no clumps
7. Add 6 mL [12 mL] buffer P2, mix by inverting the bottle vigorously 4-6 times, then incubate at room
temperature for 5 min; the bacterial lysate will become viscous as large complexes containing
bacterial proteins, broken cell walls, and denatured chromosomal DNA are precipitated from solution
when sodium ions are replaced by potassium ions
8. Add 6 mL [12 mL] chilled buffer P3, mix by inverting the bottle vigorously 4-6 times, then incubate on
ice for 15 min [20 min] and centrifuge at 3100 g for 45 min at 4oC
9. While the lysate is spinning, put together a filtration system by lining a plastic funnel with medical
gauze, and sitting it in a 50 mL tube; carefully pour the supernatant through the gauze, collecting as
much as possible, and discarding the debris in the gauze
10. Equilibrate a QIAGEN-tip 100 [QIAGEN-tip 500] by placing it over a conical flask and running 4 mL
[10 mL] buffer QBT through it and allowing it to drain completely
11. Pour the collected supernatant through the column; wash through by pouring 2 x 15 mL [2 x 30 mL]
buffer QC through the column
12. Elute the bound DNA into a fresh 50 mL tube by pouring 5 mL [15 mL] buffer QF through the column
(NB: - Buffer QN may also be used for this purpose)
13. Add 3.5 mL [10.5 mL] – i.e. 0.7 volumes – room temperature isopropanol; mix, and incubate at room
temperature for a minimum of 2 min
14. Have ready an appropriate number of 1.5 mL tubes; divide the mixture into them (1.5 mL per tube)
and centrifuge at 15000 g for 30-60 min at 4oC; carefully pour off all the supernatants
15. Using a 1000 µL filter tip, add 800 µL 70% v/v ethanol to the first tube; gently pipette up and down to
ensure that the pellet is resuspended, then transfer the contents (including the pellet) to the second
tube; repeat this process for all tubes, until all of the pellets/washings are merged in the final tube; put
this tube aside
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16. Using a fresh 1000 µL filter tip, re-wash the other tubes; add 1 mL 70% v/v ethanol to the first tube;
pipette up and down, and transfer the solution to the second tube; repeat for all of the remaining
tubes
17. Centrifuge the pellet-containing tube and the washings-tube (making sure they balance) at 15000 g
for 30-60 min at 4oC
18. Remove the supernatant, taking care not to disturb the pellet, then use a Thirsty-Stix, cotton bud or
tissue to remove as much residual ethanol as possible
19. Complete the drying of the pellet by placing it at 50-55oC in the hybridisation oven for 40-60 sec; do
not overdry!! Check for residual ethanol, and examine the pellet; when dry, it will become clear
around the edges
20. If the DNA is to be used immediately, resuspend it in molecular biology-grade H2O; for longer term
storage, use TE buffer
The resuspension volume is according to the size of the pellet, 150-200 µL for a Midi-Prep, 300-400 µL for
a Maxi-Prep; less for the pellet in the “washings” tube; it may be necessary to pipette the pellet up and
down to encourage resuspension, or to incubate it at 37oC
21. Merge the main DNA solution with that from the “washings” tube
22. Determine the concentration of the DNA using the Nanodrop spectrophotometer
23. Store at -20oC
List all resources required including plant, chemicals,
personal protective clothing and equipment, etc
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L-broth (LB) (see SWP M4)
Solutions 1, 2 and 3 (see SWP M4)
TE buffer (see SWP M4)
Appropriate selection agent for your plasmid
Agar plate(s) with colonies or a glycerol stock
Lysozyme (Sigma, cat. # L-7651; stored at -20oC) (NB: - this is an irritant)
Phenol, saturated, pH 6.6 (MP Biomedicals, cat. # 802519; stored at 4 oC) (NB: - this is a hazardous
and toxic substance)
Chloroform (BDH, cat. # 10077.6B) (NB: - this is a hazardous substance)
Absolute ethanol (Lomb, cat. # JJOOABS20) (NB: - this is a hazardous and flammable substance)
70% v/v ethanol (NB: - this is a hazardous and flammable substance)
RNase A (Sigma, cat. # R-6513; stored at -20oC; 10 mg/mL stock) (NB: - this is an irritant)
3 M sodium acetate (prepared from powder and sterilised)
Glass wool (NB: - this is a hazardous substance)
4.4 M lithium chloride (NB: - this is a hazardous substance)
Isopropanol (NB: - this is a hazardous and flammable substance)
10% sodium dodecyl sulphate (SDS) (NB: - this is a hazardous substance)
QIAGEN Plasmid Midi-Kit (cat. # 12143 or 12145)
QIAGEN Plasmid Maxi-Kit (cat. # 12163 or 12165)
Buffers P1, P2 and P3 (see SWP M4)
Buffer QBT (see SWP M4)
Buffer QC (see SWP M4)
Buffer QF (see SWP M4)
Buffer QN (see SWP M4)
Terrific broth (TB) (see SWP M4)
80% glycerol
Medical gauze
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List potential hazards and risk controls including specific
precautions required
There are several hazardous, flammable and toxic substances used in the various sections. PPE- latex
gloves, long-sleeve laboratory gowns and covered shoes, MUST be worn at all times when carrying out
any of these procedures.
Aliquots of flammable liquids such as chloroform, absolute ethanol should be used and aliquots made in
the fume hood when possible.
Extra CAUTION should be taken when handling glass wool is a respiratory hazard
When dealing with GMO, PC2 regulations has to be consulted
List emergency shutdown instructions
List clean up and waste disposal requirements
Ethanol, Chlorofom and Phenol waste has to be collected separately and collected as chemical waste
List legislation, standards and codes of practice used in
the development of the SWP
MSDS for all the listed chemicals.
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:
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