University of Oslo
Procedure with risk assessment (SOP):
Name of procedure
Written by :
IBV; Cecilie Mathiesen
1.
Saks- og dokumentnr. i ePhorte:
P-SOP-xxx-short name
Version
006
Date
16.04.2014
Page
1 av 7
Approved by:
IBV; Tor Carlsen
INTRODUCTION/PURPOSE
Standard procedure for isolation of DNA using a standard CTAB procedure.
2.
RESPONSIBILITIES AND SAFETY
See the general UiO procedure Risk management policy in laboratories for an overview of
responsibilities at UiO.
General laboratory safety applies. For more information see IBV´s HSE webpages:
http://www.mn.uio.no/ibv/english/about/hss/. Key procedures for working in the laboratories are
listed in HSE in the laboratories: http://www.mn.uio.no/ibv/english/about/hss/laboratorier/
3.
NECESSARY SAFETY EQUIPMENT
When making this solution:
 Note that no gloves protect you 100% when working with chloroform. If you spill on your
gloves you should change them immediately.
 Isopropanol (2-propanol); nitrile gloves give the best protection.
 Lab coat
 Fume hood
4.
EQUIPMENT, MATERIALS AND SOLUTIONS
Pipettes
Pipet tips, assorted sizes
Eppendorf tubes (2 and 1,5mL)
Micro Centrifuge
Mixer Mill / TissueLyzer
Crushing beads
Heating Block
milliQ water
Chemicals
CAS no
Supplier
MW=
Name
Volume
-
A4150 (Applichem
lifescience) supplied by
VWR
-
2% CTAB Buffer
~600µl per
sample
60-24-2
M3148 from Sigma
Aldrich
-
2-Mercaptoethanol
100µl per 50mL
CTAB
67-66-3
Sigma Aldrich/VWR
Chloroform
~600µl per
sample
67-63-0
Sigma Aldrich/VWR
Isopropanol
~400µl per
sample
64-17-5
Kemetyl (from
Sentrallageret)
70-75%EtOH
(diluted from 96%)
~300µl per
sample
Postal address: UiO, Department of Biosciences, P.O. Box 1066 Blindern, N-0316 Oslo
P-SOP-xxx-short name
5.
Version: 006
Page 2 of 7
PROCEDURES: DESCRIPTION OF PROCEDURE
We always wear lab coat when working in the lab.
Specific for this procedure:
 The user should also consider wearing safety goggles.
 Nitrile gloves gives the best protection. i
Remember to work in the fume hood for steps 1-7
Preparing 2%CTAB buffer with 2-mercaptoethanol
The 2% CTAB that we buy does not contain 2-Mercaptoethanol; you have to add this yourself.
Remember to wear gloves and lab coat and perform the procedures in the fume hood.
1. Pour 50mL 2%CTAB buffer into a 50mL Falcon tube.
2. Add 100µL 2-Mercaptoethanol
3. Close the lid thoroughly and invert the tube a few times to mix.
4. Wrap the tube in alu-foil and label the tube with contents, your name and date.
DNA Isolation
Notes:
 Work as sterile as possible. Change gloves whenever necessary.
 Always include a negative control.
Before starting:
Preheat the heating block to 65ºC
1.
The material is placed in 2mL eppendorf tubes and 600l of 2%CTAB buffer is to be
added.
2. Grind the material.
Usually we grind the material using the Retsch MM200 mixer mill.
Add 1-2 tungsten-carbide beads to each 2mL tube and grind for 1min at 20-30 Hz, flip the racks
and repeat for 1min.
3. Place in -80ºC freezer for minimum 10 min.
The material can stay in the -80ºC freezer overnight (or longer).
4. Incubate the mixture for 30-45 min. at 65ºC. Invert the tubes 2-3 times during
incubation.
This step lyses the cells.
5.
Allow the mixture to cool, add 600l chloroform to each tube and vortex vigorously.
6. Centrifuge the mixture for 15 min at full speed (13200 rpm).
You can prepare the tubes being used in step 7 while you are waiting. Put them in the fridge to
keep them cool once you are done adding the isopropanol.
Be careful when taking the tubes out from the centrifuge so that the two layers don’t mix again.
7.
Pipet 400 l of the upper layer (mainly water and DNA) into new 1.5mL tubes
containing 400l of cold isopropanol. Remember to mark the tubes.
Be careful not to get any of the lower layer as this includes the CTAB and the chloroform, among
other and may contaminate further steps.
8. Invert the tubes and allow the DNA to precipitate for min. 10 minutes.
You can place the tubes in the fridge (~4ºC) overnight.
9. Centrifuge at full speed for 10 minutes.
10. Carefully pour off the isopropanol and add 300l cold 70-75% ethanol. Vortex.
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P-SOP-xxx-short name
Page 3 of 7
11. Centrifuge at full speed for 2 minutes.
12. Carefully pour off the ethanol. Place to tubes on the heating block (60C) with the
lids open until everything has evaporated.
To check that there is no ethanol residue, close the tubes and flick them. If you don’t see any drops
on the walls of the tubes it is dry.
13. Add 60l of milli-Q H2O to re-suspend the DNA.
If you are purifying your DNA with any method, you should check whether to use water for this
step or some other buffer.
14. Your DNA is now isolated! Store at –20C.
Remember to label your tubes properly with one label on the side of the tube and the extr. ID
written on the lid of the tube.
6.
RISK ASSESSMENT
The likelihood is assessed by assuming the user following the precautions stated in the step by step
risk assessment (SJA) below.
6.1 List of chemicals and their H and P phrases
Chemical
2% CTAB
2-Mercaptoethanolii
Chloroformiii
Isopropanoliv
Hazard
symbol
nh
H statements
P statements
H301 + H331: Toxic if swallowed
or if inhaled
H310: Fatal in contact with skin.
H315: Causes skin irritation.
H317 May cause an allergic skin
reaction.
H318 Causes serious eye damage.
H373 May cause damage to organs
(Liver, Heart) through prolonged
or repeated exposure if swallowed.
H410 Very toxic to aquatic life
with long lasting effects.
P261: Avoid breathing vapours.
P273: Avoid release to the environment.
P280: Wear protective gloves/ eye
protection/ face protection.
P301 + P310: IF SWALLOWED:
Immediately call a POISON CENTER or
doctor/ physician.
P302 + P350: IF ON SKIN: Gently wash
with plenty of soap and water.
P305 + P351 + P338: IF IN EYES: Rinse
cautiously with water for several minutes.
Remove contact lenses, if present and easy
to do. Continue rinsing.
H302: Harmful if swallowed.
H315: Causes skin irritation.
H319: Causes serious eye
irritation.
H331: Toxic if inhaled.
H336: May cause drowsiness or
dizziness.
H351: Suspected of causing
cancer.
H361d: Suspected of damaging
the unborn child.
H372: Causes damage to organs
through prolonged or repeated
exposure.
H225 Highly flammable liquid
and vapour.
H319 Causes serious eye irritation.
H336 May cause drowsiness or
dizziness.
P261: Avoid breathing vapours.
P281: Use personal protective equipment as
required.
P305 + P351 + P338: IF IN EYES: Rinse
cautiously with water for several minutes.
Remove contact lenses, if present and easy
to do. Continue rinsing.
P311: Call a POISON CENTER or doctor/
physician.
P210 Keep away from heat, hot surfaces,
sparks, open flames and other ignition
sources. No smoking.
P261 Avoid breathing vapours.
P305 + P351 + P338 IF IN EYES: Rinse
cautiously with water for several minutes.
Remove contact lenses, if present and easy
to do. Continue rinsing
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P-SOP-xxx-short name
70-75% Ethanol
H225 Highly flammable liquid
and vapour.
H319 Causes serious eye irritation
Page 4 of 7
P210 Keep away from heat, hot surfaces,
sparks, open flames and other ignition
sources. No smoking.
P261 Avoid breathing vapours.
P305 + P351 + P338 IF IN EYES: Rinse
cautiously with water for several minutes.
Remove contact lenses, if present and easy
to do. Continue rinsing
nh= This is not classified as harmful according to the directive 67/548/EC
6.2 Risk assessment; step by step
Preparing 2%CTAB buffer with 2-mercaptoethanol
Part of procedure
Unwanted scenarios
Precautions
Emergency planning
S*K
1
Add 2% CTAB
buffer
Spillage solution
Wear gloves and
lab coat
-
1*1
2-3
Add 100µL 2Mercaptoethanol
(2-ME) and mix
Spillage solution:
H301 + H331: Toxic if
swallowed or if inhaled
H310: Fatal in contact with
skin.
H315: Causes skin
irritation.
H317 May cause an allergic
skin reaction.
H318 Causes serious eye
damage.
H373 May cause damage to
organs (Liver, Heart)
through prolonged or
repeated exposure if
swallowed.
H410 Very toxic to aquatic
life with long lasting effects
Wear gloves and
lab coat -
P305 + P351 + P338: IF IN
EYES: Rinse cautiously
with water for several
minutes. Remove contact
lenses, if present and easy
to do. Continue rinsing.
P311: Call a POISON
CENTER or doctor/
physician.
1*5
Wrap the tube in
alu-foil and label
the tube with
contents, your
name and date
-
-
-
1*1
4
Use fume hood
Tube should
be stored in
fume hood or
ventilated
chemical
cabinet prior
to use.
NOTE: If 2-ME is NOT INCLUDED in the CTAB buffer the overall risk of this procedure will be greatly reduced.
Then Chloroform will be the most hazardous chemical in this procedure and steps 1-4 will have a S*K=1*1.
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P-SOP-xxx-short name
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DNA isolation:
Part of procedure
Unwanted scenarios
Precautions
Emergency planning
S*K
1-4
To the tubes
with the
material, add
600l of 2%
CTAB w/2ME, grind
material,
freeze at -80C,
incubate.
Spillage solution:
2-ME: H301 + H331:
Toxic if swallowed or if
inhaled
H310: Fatal in contact
with skin.
H315: Causes skin
irritation.
H317 May cause an
allergic skin reaction.
H318 Causes serious eye
damage.
H373 May cause damage
to organs (Liver, Heart)
through prolonged or
repeated exposure if
swallowed.
H410 Very toxic to aquatic
life with long lasting effects
Wear gloves and
lab coat
P305 + P351 + P338:
IF IN EYES: Rinse
cautiously with water for
several minutes. Remove
contact lenses, if present
and easy to do. Continue
rinsing.
P311: Call a POISON
CENTER or doctor/
physician.
1*5
Add
chloroform,
vortex and
centrifuge
Spillage solution:
2-ME: H301 + H331:
Toxic if swallowed or if
inhaled
H310: Fatal in contact
with skin.
H315: Causes skin
irritation.
H317 May cause an
allergic skin reaction.
H318 Causes serious eye
damage.
H373 May cause damage
to organs (Liver, Heart)
through prolonged or
repeated exposure if
swallowed.
H410 Very toxic to aquatic
life with long lasting effects
Wear gloves and
lab coat -
P261: Avoid breathing
vapours
P281: Use personal
protective equipment as
required
P305 + P351 + P338:
IF IN EYES: Rinse
cautiously with water for
several minutes. Remove
contact lenses, if present
and easy to do. Continue
rinsing.
P311: Call a POISON
CENTER or doctor/
physician..
1*5
5-6
Chloroform:
H302: Harmful if
swallowed.
H315: Causes skin
irritation.
H319: Causes serious eye
irritation.
H331: Toxic if inhaled.
H336: May cause
drowsiness or dizziness.
H351: Suspected of
causing cancer.
H361d: Suspected of
damaging the unborn
child.
H372: Causes damage to
organs through prolonged
or repeated exposure.
Use fume hood
Use fume hood
IF you get
chloroform on
your gloves
change
immediately.
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P-SOP-xxx-short name
Part of procedure
Unwanted scenarios
Precautions
7-9
Add upper
layer into
isopropanol,
mix,
precipitate,
Centrifuge
2-propanol:
H225 Highly flammable
liquid and vapour.
H319 Causes serious eye
irritation.
H336 May cause
drowsiness or dizziness.
Wear gloves,
goggles and lab
coat -
Remove
isopropanol,
add 70-75%
ethanol,
centrifuge.
2-propanol: As pt 7.
Wear gloves,
goggles and lab
coat
Remove
ethanol,
evaporate, add
water, store
EtOH:
H225: Highly flammable
liquid and vapour.
H319: Causes serious eye
irritation
10-11
12-14
EtOH:
H225: Highly flammable
liquid and vapour.
H319: Causes serious eye
irritation
Use fume hood
IF you get
chloroform on
your gloves
change
immediately.
Page 6 of 7
Emergency planning
P210: Keep away from
heat, hot surfaces,
sparks, open flames and
other ignition sources.
No smoking.
P261: Avoid breathing
vapours.
P305 + P351 + P338:
IF IN EYES: Rinse
cautiously with water for
several minutes. Remove
contact lenses, if present
and easy to do. Continue
rinsing
S*K
As pt 7.
1*3
As pt 7.
1*2
1*5
Use fume hood
Wear gloves,
goggles and lab
coat
6.3 Overall risk assessment for this SOP
Risk categories
 Red: S*K=10-25 the overall risk of making this solution is unacceptable risk. Access new
precaution to reduce the risk should established.
 Yellow: S*K=4-9 the overall risk of making this solution is medium. Access new precaution
to reduce the risk should considered.
 Green: S*K=1-4 the overall risk of making this solution is fully acceptable - minimal risk.
If S*K of the step by step risk assessment falls into different categories (as listed above), the overall
risk is set to the highest S*K value.
When following this SOP, there is MEDIUM risk associated with this procedure, as some S*K values
are higher than or equal to 5.
NOTE: If 2-ME is NOT INCLUDED in the CTAB buffer the overall risk of this procedure will be
greatly reduced. Then Chloroform will be the most hazardous chemical in this procedure and steps 1-4
will have a S*K=1*1.
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P-SOP-xxx-short name
7.
Page 7 of 7
WASTE DISPOSAL
Preparing 2%CTAB buffer with 2-mercaptoethanol
Waste
Volume
Disposal method
Enviromental risk
1
-
Leave in fume hood to
evaporate.
None, since this is according
to procedure and handled by
trained staff and collected by
professionals.
Tips and gloves
contaminated with 2-ME
Dispose in hazardous waste
box.
2
Small chemical spillage
of 2-ME
0.1 – 1 ml
Clean up with paper or PigHaz mats. Allow to
evaporate in fume hood.
Dispose in hazardous waste
box.
As above
Waste
Volume
Disposal method
Enviromental risk
1
Tips and gloves
contaminated with 2-ME,
chloroform, isoporopanol
or ethanol.
-
Leave in fume hood to
evaporate.
None, since this is according
to procedure and hazardous
waste is handled by trained
staff and collected by
professionals.
2
Small chemical spillage
of 2-ME, chloroform,
isopropanol or ethanol.
0.1 – 1 ml
Clean up with paper or PigHaz mats. Allow to
evaporate in fume hood.
Dispose in hazardous waste
box.
As above
3
Chemical spillage of
isopropanol or ethanol
up to 10 ml
Clean up with paper or PigHaz mats. Allow to
evaporate in fume hood.
Dispose as normal waste.
None
4
Buffer waste from step 7
Up to 50mL
Pour in waste container
labelled chloroform waste
(halogenated
hydracarbons)
None, since this is according
to procedure and hazardous
waste is handled by trained
staff and collected by
professionals
5
Buffer waste from step 10
Up to 50mL
Pour in waste container
labelled alcohol waste
As above
6
Buffer waste from step 12
Up to 50mL
Pour in waste container
labelled alcohol waste
As above
DNA-isolation
8.
Dispose in hazardous waste
box.
REFERENCES
Guide-Gloves: http://www.guide.eu/files/pdf/GUI_Kem-EN.pdf
MSDS – 2- Mercaptoethanol
http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=NO&language=ENgeneric&productNumber=M3148&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaal
drich.com%2Fcatalog%2Fsearch%3Fterm%3DM3148%26interface%3DAll%26N%3D0%26mode%3D
match%2520partialmax%26lang%3Den%26region%3DNO%26focus%3Dproduct
iii MSDS – Chloroform:
https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=NO&language=ENgeneric&productNumber=372978&brand=SIAL&PageToGoToURL=https%3A%2F%2Fwww.sigmaald
rich.com%2Fcatalog%2Fproduct%2Fsial%2F372978%3Flang%3Den
iv MSDS – 2-propanol:
http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=NO&language=ENgeneric&productNumber=278475&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldr
ich.com%2Fcatalog%2Fproduct%2Fsial%2F278475%3Flang%3Den
i
ii