Thermo Fisher Scientific GeneCatcher gDNA Blood Kits User guide

Brand
Thermo Fisher Scientific
Model
GeneCatcher gDNA Blood Kits
Type
User guide
For Research Use Only. Not for use in diagnostic procedures.
GeneCatcher™ gDNA Blood Kits
USER GUIDE
For purification of gDNA from human blood
Catalog Numbers CS21101 and CS21110
Document Part Number 25–0828
Publication Number MAN0007154
Revision A.0
The information in this guide is subject to change without notice.
DISCLAIMER
TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0007154
Revision Date Description
A.0 27 June 2016 Replaced 24-well Ubottom deep-well plates by KingFisher™ Flex 24 Deep-
Well Plates.
— 07 September 2012 Baseline for revisions.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2016 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER 1 Product information ....................................... 5
Product description ............................................................. 5
Contents and storage ............................................................ 6
Required materials not supplied .................................................. 6
■CHAPTER 2 Isolate gDNA from 0.3–1 mL of human blood ........... 8
Procedural guidelines ........................................................... 8
Before you begin ................................................................ 8
Bind the gDNA to the magnetic beads .............................................. 8
Treat gDNA with protease ........................................................ 9
Wash gDNA on the magnetic beads ................................................ 9
Elute the gDNA ................................................................ 10
■CHAPTER 3 Isolate gDNA from 3–10 mL of human blood ........... 12
Procedural guidelines .......................................................... 12
Before you begin ............................................................... 12
Bind the gDNA to the magnetic beads ............................................. 13
Treat gDNA with protease ....................................................... 13
Wash gDNA on the magnetic beads ............................................... 14
Elute the gDNA ................................................................ 15
■CHAPTER 4 Determine the gDNA yield ............................... 16
Determine the gDNA yield using UV absorbance ................................... 16
Determination of the gDNA yield using the Quant-iT™ kits ........................... 16
■APPENDIX A Troubleshooting ......................................... 17
■APPENDIX B Safety ..................................................... 18
Chemical safety ................................................................ 19
Biological hazard safety ......................................................... 20
GeneCatcher™ gDNA Blood Kits User Guide
3
■Documentation and support ............................................. 21
Customer and technical support ................................................. 21
Limited product warranty ....................................................... 21
Contents
4
GeneCatcher™ gDNA Blood Kits User Guide
Product information
IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
Product description
The GeneCatcher™ gDNA Blood Kits allow rapid and ecient extraction of genomic
DNA (gDNA) from human blood including archived or poorly stored blood samples.
Genomic DNA is extracted from blood samples Using the GeneCatcher™ magnetic
bead technology, the kits provides high purity and high yield of gDNA appropriate
for use in any downstream applications including PCR and restriction enzyme
digestion.
The GeneCatcher™ gDNA Blood Kits are optimized to purify gDNA from various
types of human blood samples including:
• Fresh, whole blood
• Blood collected in the presence of anti-coagulants such as EDTA, heparin, or
citrate
• Frozen blood samples or blood samples exposed to repeated freeze-thaw cycles
• Old, archived blood samples and degraded samples
This guide described 2 methods for gDNA isolation optimized for small and large
sample volumes.
Sample volume Cat. No. Methods
0.3–1 mL CS21101 Chapter 2, “Isolate gDNA from 0.3–1 mL of
human blood“
3–10 mL CS21110 Chapter 3, “Isolate gDNA from 3–10 mL of
human blood“
1
GeneCatcher™ gDNA Blood Kits User Guide
5
Contents and storage
Cat. No. CS21101 provides sucient reagents for 96 purications using ≤1 mL of
blood. Cat. No. CS21110 provides sucient reagents for 66 purications using 3 mL of
blood or 20 purications using 10 mL of blood
Table 1 GeneCatcher™ gDNA Blood Kits (Cat. Nos. CS21101 and CS21110)
Contents Cat. No.
CS21101
Cat. No.
CS21110 Storage
GeneCatcher™ Magnetic Beads,
25 mg/mL 6 mL 3 mL
15–30°C
GeneCatcher™ Lysis Buffer (L13) 480 mL 1000 mL
Protease, 25 mg/mL 1 mL 2.7 mL 2–8°C
Protease Buffer 50 mL 200 mL
15–30°CGeneCatcher™ Wash Buffer (W12) 55 mL 55 mL
GeneCatcher™ Elution Buffer (E5) 55 mL 55 mL
Required materials not supplied
Unless otherwise indicated, all materials are available through thermosher.com.
MLS: Fisher Scientic (www.sherscientic.com) or other major laboratory supplier.
Table 2 Materials required for gDNA isolation (all sample sizes)
Item Source
Equipment
Heat block or water bath at 65°C, 55°C, and 90°C MLS
Adjustable micropipettors MLS
Pipettes, sterile MLS
Consumables
Aerosol-resistant pipette tips MLS
Reagents
Isopropanol, 100% MLS
(Optional)
Quant-iT™ DNA Assay Kit, High Sensitivity Q33120
(Optional)
Quant-iT™ DNA Assay Kit, Broad-Range Q33130
(Optional)
Quant-iT™ PicoGreen™ dsDNA Assay P7589
Chapter 1 Product information
Contents and storage
1
6
GeneCatcher™ gDNA Blood Kits User Guide
Table 3 Additional materials required for gDNA isolation (0.3–1 mL of blood)
Item Source
Equipment
24-Well Magnetic Separator CS15024
(Optional)
Plate shaker with a minimum orbit of 2 mm
operating at 800–1000 rpm
MLS
Plates
KingFisher™ Flex 24 Deep-Well Plates 95040480
Table 4 Additional materials required for gDNA isolation (3–10 mL of blood)
Item Source
Equipment
50-mL Tube Magnetic Separator CS15050
Vortex MLS
Tubes
50-mL tubes, sterile MLS
Chapter 1 Product information
Required materials not supplied
1
GeneCatcher™ gDNA Blood Kits User Guide
7
Isolate gDNA from 0.3–1 mL of
human blood
Procedural guidelines
• Perform all steps at room temperature (20–25°C) unless otherwise noted.
• Volumes for reagent mixes are given per sample. We recommend that you
prepare master mixes for larger sample numbers. To calculate volumes for master
mixes, refer to the per-well volume and add 5–10% overage.
• Maintain a sterile environment when handling DNA to avoid any contaminations
from DNases.
• Do not allow the magnetic beads to dry as drying reduces the beads eciency.
• Do not freeze the magnetic beads as freezing damages the beads.
•Mix the magnetic beads by gentle agitation using a plate shaker set to low speeds
(800–1000 rpm).
• When mixing samples by pipeing up and down, avoid creating bubbles.
• To aspirate the supernatant after bead washing, place the pipee tip away from
the beads by angling the pipee such that the tip is pointed away from the pellet
and carefully remove the supernatant without disturbing or removing any beads.
• To obtain best results, elute the gDNA using the provide Elution Buer (E5). Do
not use water for elution.
Before you begin
• Resuspend well the magnetic beads by vortexing.
• Set a heat block or water bath at 65°C.
• Mix well the blood samples.
Bind the gDNA to the magnetic beads
1. Add 60 µL of magnetic beads to the wells of a 24-well plate.
2. Add 2.5 mL of Lysis Buer (L13) to the wells.
3. Mix well by gently swirling or by gently agitation using a plate shaker.
4. Add 0.3–1 mL of the blood samples.
2
8
GeneCatcher™ gDNA Blood Kits User Guide
5. Mix well by gently swirling or by gently agitation using a plate shaker.
6. Incubate for 5 minutes to allow the DNA to bind to the magnetic beads, agitating
occasionally by gently swirling the plate.
7. Place the plate on the Magnetic Separator for 3 minutes.
8. Without removing the plate from the Magnetic Separator, carefully remove and
discard the supernatant .
9. Remove the plate from the Magnetic Separator.
10. Add 2.5 mL of Lysis Buer (L13) to the wells.
11. Mix well by gently swirling or by gently agitation using a plate shaker.
12. Place the plate on the Magnetic Separator for 1 minute.
13. Without removing the plate from the Magnetic Separator, carefully remove and
discard the supernatant .
Treat gDNA with protease
1. Remove the plate from the Magnetic Separator.
2. Add 0.5 mL of Protease Buer and 10 µL of Protease to the sample.
3. Mix well by gently swirling or by gently agitation using a plate shaker until the
magnetic bead pellet is completely dispersed.
If the magnetic bead pellet does not easily disperse, warm the plate at 65°C for
5 minutes and pipet up and down gently to disperse the pellet without forming
any bubbles.
4. Incubate the plate at 65°C for 10 minutes.
5. Cool the plate down to room temperature (~10–20 minutes).
6. Mix well by gently swirling or by gently agitation using a plate shaker.
Wash gDNA on the magnetic beads
1. Add 0.5 mL of 100% isopropanol to the samples.
2. Mix well by gently swirling or by gently agitation using a plate shaker until a
visible aggregate is formed in the sample wells.
Absence of aggregate after 2–3 minutes indicated very low DNA levels in the
sample.
The supernatant should be clear, olive green color.
3. Place the plate on the Magnetic Separator for 30 seconds to 1 minute.
Chapter 2 Isolate gDNA from 0.3–1 mL of human blood
Treat gDNA with protease
2
GeneCatcher™ gDNA Blood Kits User Guide
9
4. Without removing the plate from the Magnetic Separator, carefully remove and
discard the supernatant .
5. Remove the plate from the Magnetic Separator.
6. Add 1 mL of 50% (v/v) isopropanol to the samples.
7. Mix well by gently swirling or by gently agitation using a plate shaker for
15 seconds.
8. Place the plate on the Magnetic Separator for 30 seconds.
9. Without removing the plate from the Magnetic Separator, carefully remove and
discard the supernatant .
10. Add 150 µL of Wash Buer (W12) to the sides of the sample wells opposite to the
bead pellet to ensure that the pellet is not disturbed.
11. Without removing the plate from the Magnetic Separator, carefully remove and
discard the supernatant.
12. Repeat step 10-step 11.
Elute the gDNA
1. Remove the plate from the Magnetic Separator.
2. Add 250 µL of Elution Buer (E5) to the samples.
3. Mix well by gently swirling or by gently agitation using a plate shaker.
4. Incubate at 65°C for 30 minutes.
For high DNA content, incubate samples for an additional 30 minutes.
5. Mix well by gently swirling or by gently agitation using a plate shaker.
If the pellet is not completely dispersed, use a clean, sterile pipee tip to
dispersed the pellet without forming bubbles. For high DNA content, incubate
the tube over night and use a clean, sterile pipee tip to dispersed the pellet
without forming bubbles.
If the supernatant is too viscous, add an additional 150–250 µL of Elution Buer
(E5) and incubate the sample over night.
6. Place the plate on the Magnetic Separator until the supernatant is clear and
colorless (~15 minutes to 1 hour).
Viscous samples require longer time.
7. Without removing the plate from the Magnetic Separator, carefully remove the
DNA-containing supernatant and transfer to a sterile tube.
Chapter 2 Isolate gDNA from 0.3–1 mL of human blood
Elute the gDNA
2
10
GeneCatcher™ gDNA Blood Kits User Guide
The puried gDNA is ready for immediate use. Alternatively, carefully transfer the
supernatant to a new tube and store:
• At 4°C for immediate use.
• At –20°C in aliquots for long-term storage.
Chapter 2 Isolate gDNA from 0.3–1 mL of human blood
Elute the gDNA
2
GeneCatcher™ gDNA Blood Kits User Guide
11
Isolate gDNA from 3–10 mL of
human blood
Procedural guidelines
• Perform all steps at room temperature (20–25°C) unless otherwise noted.
• Volumes for reagent mixes are given per sample. We recommend that you
prepare master mixes for larger sample numbers. To calculate volumes for master
mixes, refer to the per-well volume and add 5–10% overage.
• Maintain a sterile environment when handling DNA to avoid any contaminations
from DNases.
• Do not allow the magnetic beads to dry as drying reduces the beads eciency.
• Do not freeze the magnetic beads as freezing damages the beads.
•Mix the magnetic beads by gently inverting the tube or pipeing up and down.
• When mixing samples by pipeing up and down, avoid creating bubbles.
• To aspirate the supernatant after bead washing, place the pipee tip away from
the beads by angling the pipee such that the tip is pointed away from the pellet
and carefully remove the supernatant without disturbing or removing any beads.
• To obtain best results, elute the gDNA using the provide Elution Buer (E5). Do
not use water for elution.
Before you begin
• Resuspend well the magnetic beads by vortexing.
• Set a heat block or water bath at 65°C.
• Mix well the blood samples.
3
12
GeneCatcher™ gDNA Blood Kits User Guide
Bind the gDNA to the magnetic beads
1. Add magnetic beads and Lysis Buer (L13) to a 50-mL sterile tube according to
the following table.
Reagents
Sample volume
3 mL 4 mL 5 mL 6 mL 7 mL 8 mL 9 mL 10 mL
Magnetic
beads 45 µL 60 µL 75 µL 90 µL 105 µL 120 µL 135 µL 150 µL
Lysis Buffer
(L13) 9 mL 12 mL 15 mL 18 mL 21 mL 24 mL 27 mL 30 mL
2. Mix well by gently by gently inverting the tube.
3. Add 3–10 mL of the blood samples.
4. Mix well by gently by gently inverting the tube.
5. Incubate for 5 minutes to allow the DNA to bind to the magnetic beads, agitating
occasionally by gently inverting the tube.
6. Place the tube on the 50-mL Tube Magnetic Separator for 3 minutes.
7. Without removing the plate from the Magnetic Separator, carefully remove and
discard the supernatant .
8. Remove the tube from the Magnetic Separator.
9. Add 5 mL of Lysis Buer (L13) to the tube.
10. Mix well by gently inverting the tube.
11. Place the plate on the Magnetic Separator for 20 seconds.
12. Without removing the tube from the Magnetic Separator, carefully remove and
discard the supernatant .
Treat gDNA with protease
1. Remove the tube from the Magnetic Separator.
2. Add Protease Buer and Protease to the sample according to the following table.
Reagents
Sample volume
3 mL 4 mL 5 mL 6 mL 7 mL 8 mL 9 mL 10 mL
Protease
Buffer 3 mL 3 mL 3 mL 3 mL 3.5 mL 4 mL 4.5 mL 5 mL
Protease 40 µL 40 µL 40 µL 40 µL 40 µL 40 µL 40 µL 40 µL
Chapter 3 Isolate gDNA from 3–10 mL of human blood
Bind the gDNA to the magnetic beads
3
GeneCatcher™ gDNA Blood Kits User Guide
13
3. Incubate the tube at 65°C for 10 minutes.
4. Cool the tube down to room temperature (~10–20 minutes).
5. Mix well by gently inverting the tube.
Wash gDNA on the magnetic beads
1. Add 100% isopropanol to the samples according to the following table.
Reagents
Sample volume
3 mL 4 mL 5 mL 6 mL 7 mL 8 mL 9 mL 10 mL
100%
Isopropanol 3 mL 3 mL 3 mL 3 mL 3.5 mL 4 mL 4.5 mL 5 mL
2. Mix well by gently by gently inverting the tube until a visible aggregate is
formed in the sample wells.
Absence of aggregate after 2–3 minutes indicated very low DNA levels in the
sample.
The supernatant should be clear, olive green color.
3. Place the tube on the Magnetic Separator for 30 seconds to 1 minute.
4. Without removing the tube from the Magnetic Separator, carefully remove and
discard the supernatant .
5. Remove the tube from the Magnetic Separator.
6. Add 3 mL of 50% (v/v) isopropanol to the samples.
7. Mix well by gently inverting the tube 5 times.
8. Place the tube on the Magnetic Separator for 30 seconds.
9. Without removing the tube from the Magnetic Separator, carefully remove and
discard the supernatant .
10. Keep the tube on the Magnetic Separator for an additional 1 minute to allow any
remaining liquid to sele at the boom of the tube.
11. Remove and discard the liquid using a pipee.
12. Add 250 µL of Wash Buer (W12) to the sides of the tube opposite to the bead
pellet to ensure that the pellet is not disturbed.
13. Without removing the tube from the Magnetic Separator, carefully remove and
discard the supernatant.
14. Repeat step 12-step 13.
Chapter 3 Isolate gDNA from 3–10 mL of human blood
Wash gDNA on the magnetic beads
3
14
GeneCatcher™ gDNA Blood Kits User Guide
Elute the gDNA
1. Remove the tube from the Magnetic Separator.
2. Add 1 mL of Elution Buer (E5) to the samples.
3. Mix well by gently inverting the tube.
4. Incubate at 65°C for 1 hour.
For high DNA content, incubate samples for an additional 30 minutes.
5. Mix well by gently inverting the tube.
If the pellet is not completely dispersed, use a clean, sterile pipee tip to
dispersed the pellet without forming bubbles. For high DNA content, incubate
the tube over night and use a clean, sterile pipee tip to dispersed the pellet
without forming bubbles.
If the supernatant is too viscous, add an additional 0.5–1 mL of Elution Buer
(E5) and incubate the sample over night.
6. Place the tube on the Magnetic Separator until the supernatant is clear and
colorless (~15 minutes to 1 hour).
Viscous samples require longer time.
7. Without removing the tube from the Magnetic Separator, carefully remove the
DNA-containing supernatant and transfer to a sterile tube.
If the supernatant is discolored, repeat step 6-step 7.
The puried gDNA is ready for immediate use. Alternatively, carefully transfer the
supernatant to a new tube and store:
•At 4°C for immediate use.
• At –20°C in aliquots for long-term storage.
Chapter 3 Isolate gDNA from 3–10 mL of human blood
Elute the gDNA
3
GeneCatcher™ gDNA Blood Kits User Guide
15
Determine the gDNA yield
Perform gDNA quantitation using UV absorbance at 260 nm or Quant-iT™ kits.
Determine the gDNA yield using UV absorbance
1. Prepare a dilution of gDNA solution in 10 mM Tris-HCl, pH 7.5, then mix well.
2. Measure the absorbance at 260 nm (A260) of the dilution in a spectrophotometer
using a cuvee with an optical path length of 1 cm and blanked against 10 mM
Tris-HCl, pH 7.5.
3. Calculate the concentration of gDNA using the following formula.
DNA (µg/mL) = A260 × 50 × dilution factor
For DNA, A260 = 1 for a 50 µg/mL solution measured in a cuvee with an optical
path length of 1 cm.
Determination of the gDNA yield using the Quant-iT™ kits
The Quant-iT™ kits provide a rapid, sensitive, and specic uorescent method for
dsDNA quantitation. The kits contain a state-of-the-art quantitation reagent, DNA
standards for standard curve, and a pre-made buer to allow uorescent DNA
quantitation using standard uorescent microplate readers or uorometers.
4
16
GeneCatcher™ gDNA Blood Kits User Guide
Troubleshooting
Observation Possible cause Recommended action
The gDNA yield is low The reagent amount is
incorrect.
Use the appropriate amounts of magnetic
beads and reagents based on the amount of
starting materials.
The magnetic beads were
improperly handled.
Vortex the tube containing the magnetic beads
to fully resuspend the beads in solution prior to
use.
Ensure that the beads are mixed occasionally
during all incubation steps.
The bead pellet was disturbed
or lost during the binding or
washing steps.
Keep the sample on the Magnetic Separator
when removing the supernatant during the
binding and washing steps.
Remove carefully the supernatant without
disturbing the bead pellet by angling the
pipette tip away from the pellet.
The elution conditions are
incorrect.
After adding the Elution Buffer (E5) to the
sample, fully resuspend the magnetic beads
before incubation.
Perform elution at 65°C.
Do not use water to elute DNA.
No DNA was recovered Water was used for elution. Do not use water to elute DNA.
The elution buffer must have a pH of 8.5–9.0 or
the DNA remains bound to the magnetic beads.
The magnetic beads were
stored or handled improperly.
Store magnetic beads at room temperature.
Freezing the beads irreparably damaged them.
Ensure that the magnetic beads are always in
solution and are not dried. Dried beads cannot
bind DNA.
The DNA is sheared. Perform all mixing steps gently, using a plate
shaker for 24-well plate or inverting the 50-mL
tube.
A
GeneCatcher™ gDNA Blood Kits User Guide
17
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specied
in the user documentation may result in personal injury or damage to the
instrument or device. Ensure that anyone using this product has received
instructions in general safety practices for laboratories and the safety
information provided in this document.
·Before using an instrument or device, read and understand the safety
information provided in the user documentation provided by the
manufacturer of the instrument or device.
·Before handling chemicals, read and understand all applicable Safety Data
Sheets (SDSs) and use appropriate personal protective equipment (gloves,
gowns, eye protection, etc). To obtain SDSs, see the “Documentation and
Support” section in this document.
B
18
GeneCatcher™ gDNA Blood Kits User Guide
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,
ensure laboratory personnel read and practice the general safety guidelines for
chemical usage, storage, and waste provided below. Consult the relevant SDS
for specic precautions and instructions:
·Read and understand the Safety Data Sheets (SDSs) provided by the
chemical manufacturer before you store, handle, or work with any chemicals
or hazardous materials. To obtain SDSs, see the “Documentation and
Support” section in this document.
·Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing).
·Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood).
·Check regularly for chemical leaks or spills. If a leak or spill occurs, follow
the manufacturer's cleanup procedures as recommended in the SDS.
·Handle chemical wastes in a fume hood.
·Ensure use of primary and secondary waste containers. (A primary waste
container holds the immediate waste. A secondary container contains spills
or leaks from the primary container. Both containers must be compatible
with the waste material and meet federal, state, and local requirements for
container storage.)
·After emptying a waste container, seal it with the cap provided.
·Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
·Ensure that the waste is stored, transferred, transported, and disposed of
according to all local, state/provincial, and/or national regulations.
·IMPORTANT! Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.
Appendix B Safety
Chemical safety
B
GeneCatcher™ gDNA Blood Kits User Guide
19
Biological hazard safety
WARNING! BIOHAZARD. Biological samples such as tissues, body uids,
infectious agents, and blood of humans and other animals have the potential to
transmit infectious diseases. Conduct all work in properly equipped facilities
with the appropriate safety equipment (for example, physical containment
devices). Safety equipment can also include items for personal protection, such
as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety
glasses, or goggles. Individuals should be trained according to applicable
regulatory and company/ institution requirements before working with
potentially biohazardous materials. Follow all applicable local, state/provincial,
and/or national regulations. The following references provide general
guidelines when handling biological samples in laboratory environment.
·U.S. Department of Health and Human Services, Biosafety in Microbiological
and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)
21-1112, Revised December 2009; found at:
www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf
·World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
Appendix B Safety
Biological hazard safety
B
20
GeneCatcher™ gDNA Blood Kits User Guide
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Thermo Fisher Scientific GeneCatcher gDNA Blood Kits User guide

Brand
Thermo Fisher Scientific
Model
GeneCatcher gDNA Blood Kits
Type
User guide
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