Standard Operating Procedure: NRL003
Extraction of Anisakis DNA using the DNA IQ
Tissue and Hair Extraction method
(DOC 28 – Revision 1)
Centre for Environment, Fisheries and Aquaculture Science,
Weymouth Laboratory, Weymouth, Dorset, UK, DT4 8UB
Tel +44 1305 206600, Fax : +44 1305 206601.
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NRL003 Extraction of Anisakis DNA using the DNA IQ Tissue and Hair Extraction method – DOC28
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NRL003 Extraction of Anisakis DNA using the DNA IQ Tissue and Hair Extraction method – DOC28
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Version Control
Submitted to:
Date submitted
Project manager
Document compiled by:
Quality control by:
Approved by and date:
Version:
Steve Feist
8/1/2015
Steve Feist
Rose Kerr
Steve Feist
Steve Feist 8/1/2015
1.3
Author
Date
Comment
Version
Rose Kerr
8/1/2015
DRAFT
1.1
Steve Feist
8/1/2015
Approved
1.2
Tom Hill
8/10/2014
Saved to contracts drive
1.3
Tom Hill
25/6/2015
Version control added and uploaded to
QPulse as ‘DOC28 - Revision 1’
DOC28 Revision 1
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NRL003 Extraction of Anisakis DNA using the DNA IQ Tissue and Hair Extraction method – DOC28
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Introduction
The Tissue and Hair extraction kit for use with the DNA IQ system is used for the extraction of total DNA.
Specifically the use of Dithiothreitol (DTT) in the IPD (Incubation buffer, Proteinase K and DTT) mix in the
initial incubation reduces disulfide bonds to dithiols, which results in the external proteins breaking down. An
Anisakid worm will be digested into a liquid at this stage. Further lysis of internal proteins occurs via the use of
excess Proteinase K in the lysis buffer stage. This results in a more complete release of total DNA into solution
which is precipitated using Isopropyl alcohol and ETOH washing stages and finally eluted into molecular grade
water.
Scope
This standard operating procedure covers the specific lysis and extraction of Anisakis ssp DNA only.
Equipment
Nitrile gloves
Laboratory coat
Variable volume pipettors p10, p100, p200, p1000 and associated tips (eg Rainin pipet lite XLS)
Plastic autoclaved racks
Mini centrifuge (eg Stuart Spinner SCF1 Bibby Scientific)
Vortexer (eg Stuart SA8 Bibby Scientific)
Magnetic stand
Waste bag
500ml beaker
0.5ml PCR tubes
1.5ml microfuge tubes
Universals
Fume cupboard
Extraction cabinet (Labconco purifier PCR enclosure)
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NRL003 Extraction of Anisakis DNA using the DNA IQ Tissue and Hair Extraction method – DOC28
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Fridge in room 213(Moff 7)
(small) Freezer room 213
Variable temperature heat block
Centrifuge (Heraeus Biofuge Pico)
Chemicals
99% absolute ETOH
70% ETOH in a spray bottle
1% Virkon (1 tablet in 500mls H20) in a spray bottle
Molecular grade H20
Isopropyl alcohol
Tissue and Hair Extraction Kit (for use with DNA IQ). Part# TB307 containing:
100mg Proteinase K powder
5g DTT powder
2 x Wash buffer concentrate
Lysis buffer
Incubation buffer
Resin
Elution buffer
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NRL003 Extraction of Anisakis DNA using the DNA IQ Tissue and Hair Extraction method – DOC28
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Method
The following procedures take place in room 213. A laboratory coat and gloves must be won for all
stages.
Prior to undertaking the DNA extraction the following buffers must be made up: Proteinase K, DTT,
IB/ProtK/DTT mix, Wash buffer, Lysis buffer.
Clean cabinet (A, B or C) and associated pipettes, mini centrifuge and vortexer using 1% Virkon
solution followed by 70% ETOH with paper towel. UV the cleaned cabinet for 5 minutes. After the
UV has switched off place in the cabinet a waste bag, gloves, pipette tips (p10, p200, p1000), and
switch on the UV for 5 minutes.
To make the Proteinase K solution:
Pipette 5.5ml of Incubation Buffer into 100mg Proteinase K powder, vortex to dissolve, and pipette
into 100µl volumes. Store the labelled tubes at -20oC. The concentration of each aliquot of Proteinase
K is 18mg/ml.
To make the DTT, in a fume cupboard pipette 32.4ml of molecular grade water into the 5g of DTT
powder and shake until dissolved. Pipette 250µl volumes into 0.5ml labelled microfuge tubes and
freeze at -20oC.
To make the IPD buffer (IB / Proteinase K / DTT), the following liquids were mixed in the following
proportions: 8 parts Incubation buffer (IB) (eg 800µl), 1 part Proteinase K (eg 100µl) and 1 part DTT
(eg 100µl) in a 1.5ml microfuge tube. This mix must be made up freshly prior to each use.
To make the Wash Buffer pipette 15ml of Isopropyl alcohol and 15ml of 99% absolute ETOH into the
2 x Wash Buffer concentrate and mix and stored at room temperature.
To make the Lysis Buffer, pipette 1µl of DTT per 100µl of Lysis Buffer. The buffer can then be
stored at room temperature for 1 month.
Clean cabinet (A, B or C) and associated pipettes, mini centrifuge and vortexer using a 1% Virkon
solution followed by 70% ETOH with paper towel. UV the cleaned cabinet for 5 minutes. After the
UV has switched off place in the cabinet a waste bag (in the 500ml beaker), gloves, pipette tips (p10,
p200, p1000), labelled 1.5ml microfuge tube (one for each sample), autoclaved tube rack containing
labelled 0.5ml PCR tubes, one for each sample. Also place in the cabinet a universal container
labelled ‘waste’ and the magnetic stand. Switch the UV back on for a further 5 minutes. Take the
microfuge tube (s) containing the worm (s) from the fridge and place in the cabinet. Pipette off the
ETOH from the worm into the waste universal container. Using a pipette tip flick the worm into the
labelled 1.5ml microfuge tube. Pipette 100µl of IPD buffer into the microfuge tube containing the
worm, and incubate at 55oC, in the heat block in 213, for 2 hours, vortexing every 30 minutes.
Centrifuge the sample briefly and pipette 200µl of Lysis Buffer into the sample. Then pipette 10µl of
thoroughly vortexed resin. Incubate the sample for 10 minutes at 25oC on the heat block, vortexing
regularly. Place the sample in the magnetic stand and once the resin has separated out from the liquid
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NRL003 Extraction of Anisakis DNA using the DNA IQ Tissue and Hair Extraction method – DOC28
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and stuck to the side of the tube pipette off the Lysis Buffer. Add another 100µl Lysis Buffer to the
microfuge tube and remove from the magnetic stand. Vortex the sample briefly, place the microfuge
tube containing the sample back in the magnetic stand, wait for the resin to stick to the magnet and
pipette off the Lysis buffer as before. Remove the microfuge tube from the magnetic stand. Add 100µl
of Wash Buffer and vortex the sample to resuspend the resin, place the microfuge tube back onto the
magnetic stand and wait for the resin to stick to the side of the tube. Pipette off the buffer, place the
tube back into the stand and repeat the wash buffer steps a further two times. After the last wash leave
the tube in the magnetic stand with the lids open to air dry for 15 minutes. Remove the microfuge tube
from the stand and pipette 25µl of Elution Buffer into the microfuge tube and incubate the sample at
65oC for 5 minutes with vortexing. Place the microfuge tube into the magnetic stand and wait for the
resin to stick to the side of the tube. Using a P200 pipette, remove the 25µl eluate from the tube and
pipette into a labelled 0.5ml PCR tube and store until required in the -20oC freezer.
Return all the items left in the cabinet to their storage locations and re clean the cabinet with 1%
Virkon followed by 70% ETOH.
Review
Yearly.
References
Tissue and Hair Extraction Kit (for use with DNA IQ). Part# TB307 Promega technical bulletin.
Revised 6/12.
DNA IQ system small sample casework protocol. Part# TB296 promega technical bulletin. Revised
11/13.
Identification of Anisakidae larvae at the species level by multiplex PCR. European Union Reference
Laboratory for Parasites. Department of Infectious, Parasitic and Immunomediated diseases. Unit of
Gastroenteric and Tissue Parasitic Diseases. Istituto Superiore di Sanita.
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NRL003 Extraction of Anisakis DNA using the DNA IQ Tissue and Hair Extraction method – DOC28
Revision 1
Printed versions of this document are uncontrolled unless signed by the Quality Department
Page 8 of 8
NRL003 Extraction of Anisakis DNA using the DNA IQ Tissue and Hair Extraction method – DOC28
Revision 1
Printed versions of this document are uncontrolled unless signed by the Quality Department